Posts Tagged ‘chemotherapeutic toxicity’

Are Cyclin D and cdk Inhibitors A Good Target for Chemotherapy?


Curator: Stephen J. Williams, Ph.D.

UPDATED 7/12/2022

see below for great review



CDK4 and CDK6 kinases: From basic science to cancer therapy

14 Jan 2022
Vol 375Issue 6577

Targeting cyclin-dependent kinases

Cyclin-dependent kinases (CDKs), in complex with their cyclin partners, modulate the transition through phases of the cell division cycle. Cyclin D–CDK complexes are important in cancer progression, especially for certain types of breast cancer. Fassl et al. discuss advances in understanding the biology of cyclin D–CDK complexes that have led to new concepts about how drugs that target these complexes induce cancer cell cytostasis and suggest possible combinations to widen the types of cancer that can be treated. They also discuss progress in overcoming resistance to cyclin D–CDK inhibitors and their possible application to diseases beyond cancer. —GKA

Structured Abstract


Cyclins and cyclin-dependent kinases (CDKs) drive cell division. Of particular importance to the cancer field are D-cyclins, which activate CDK4 and CDK6. In normal cells, the activity of cyclin D–CDK4/6 is controlled by the extracellular pro-proliferative or inhibitory signals. By contrast, in many cancers, cyclin D–CDK4/6 kinases are hyperactivated and become independent of mitogenic stimulation, thereby driving uncontrolled tumor cell proliferation. Mouse genetic experiments established that cyclin D–CDK4/6 kinases are essential for growth of many tumor types, and they represent potential therapeutic targets. Genetic and cell culture studies documented the dependence of breast cancer cells on CDK4/6. Chemical CDK4/6 inhibitors were synthesized and tested in preclinical studies. Introduction of these compounds to the clinic represented a breakthrough in breast cancer treatment and will likely have a major impact on the treatment of many other tumor types.


Small-molecule CDK4/6 inhibitors (palbociclib, ribociclib, abemaciclib) showed impressive results in clinical trials for patients with hormone receptor–positive breast cancers. Addition of CDK4/6 inhibitors to standard endocrine therapy substantially extended median progression-free survival and prolonged median overall survival. Consequently, all three CDK4/6 inhibitors have been approved for treatment of women with advanced or metastatic hormone receptor–positive breast cancers. In the past few years, the renewed interest in CDK4/6 biology has yielded several surprising discoveries. The emerging concept is that CDK4/6 kinases regulate a much wider set of cellular functions than anticipated. Consequently, CDK4/6 inhibitors, beyond inhibiting tumor cell proliferation, affect tumor cells and the tumor environment through mechanisms that are only beginning to be elucidated. For example, inhibition of CDK4/6 affects antitumor immunity acting both on tumor cells and on the host immune system. CDK4/6 inhibitors were shown to enhance the efficacy of immune checkpoint blockade in preclinical mouse cancer models. These new concepts are now being tested in clinical trials.


Palbociclib, ribociclib, and abemaciclib are being tested in more than 300 clinical trials for more than 50 tumor types. These trials evaluate CDK4/6 inhibitors in combination with a wide range of therapeutic compounds that target other cancer-relevant pathways. Several other combination treatments were shown to be efficacious in preclinical studies and will enter clinical trials soon. Another CDK4/6 inhibitor, trilaciclib, is being tested for its ability to shield normal cells of the host from cytotoxic effects of chemotherapy. New CDK4/6 inhibitors have been developed and are being assessed in preclinical and clinical trials. The major impediment in the therapeutic use of CDK4/6 inhibitors is that patients who initially respond to treatment often develop resistance and eventually succumb to the disease. Moreover, a substantial fraction of tumors show preexisting, intrinsic resistance to CDK4/6 inhibitors. One of the main challenges will be to elucidate the full range of resistance mechanisms. Even with the current, limited knowledge, one can envisage the principles of new, improved approaches to overcome known resistance mechanisms. Another largely unexplored area for future study is the possible involvement of CDK4/6 in other pathologic states beyond cancer. This will be the subject of intense studies, and it may extend the utility of CDK4/6 inhibitors to the treatment of other diseases.
Targeting cyclin D–CDK4/6 for cancer treatment.
D-cyclins (CycD) activate CDK4 and CDK6 in G1 phase of the cell cycle and promote cell cycle progression by phosphorylating the retinoblastoma protein RB1. RB1 inhibits E2F transcription factors; phosphorylation of RB1 activates E2F-driven transcription. In many cancers, CycD-CDK4/6 is constitutively activated and drives uncontrolled cell proliferation. The development of small-molecule CDK4/6 inhibitors provided a therapeutic tool to repress constitutive CycD-CDK4/6 activity and to inhibit cancer cell proliferation. As with several targeted therapies, tumors eventually develop resistance and resume cell proliferation despite CDK4/6 inhibition. New combination treatments, involving CDK4/6 inhibitors plus inhibition of other pathways, are being tested in the clinic to delay or overcome the resistance.



Cyclin-dependent kinases 4 and 6 (CDK4 and CDK6) and their activating partners, D-type cyclins, link the extracellular environment with the core cell cycle machinery. Constitutive activation of cyclin D–CDK4/6 represents the driving force of tumorigenesis in several cancer types. Small-molecule inhibitors of CDK4/6 have been used with great success in the treatment of hormone receptor–positive breast cancers and are in clinical trials for many other tumor types. Unexpectedly, recent work indicates that inhibition of CDK4/6 affects a wide range of cellular functions such as tumor cell metabolism and antitumor immunity. We discuss how recent advances in understanding CDK4/6 biology are opening new avenues for the future use of cyclin D–CDK4/6 inhibitors in cancer treatment.
Cyclin D1, the activator of CDK4 and CDK6, was discovered in the early 1990s (12). The role of cyclin D1 in oncogenesis was already evident at the time of its cloning, as it was also identified as the protein product of the PRAD1 oncogene, which is rearranged and overexpressed in parathyroid adenomas (3), and of the BCL1 oncogene, which is rearranged in B-lymphocytic malignancies (4). Subsequently, the remaining two D-type cyclins, D2 and D3, were discovered on the basis of their homology to cyclin D1 (1).
Cyclins serve as regulatory subunits of cyclin-dependent kinases (CDKs) (5). Shortly after the discovery of D-cyclins, CDK4 and CDK6 were identified as their kinase partners (6). Mouse gene knockout studies revealed that CDK4 and CDK6 play redundant roles in development, and combined ablation of CDK4 and CDK6 was found to result in embryonic lethality (7). The essentially identical phenotype was seen in cyclin D–knockout mice, thereby confirming the role of D-cyclins as CDK4/6 activators in vivo (8). Surprisingly, these analyses revealed that many normal nontransformed mammalian cell types can proliferate without any cyclin D–CDK4/6 activity (78).
CDK4 and CDK6 are expressed at constant levels throughout the cell cycle. By contrast, D-cyclins are labile proteins that are transcriptionally induced upon stimulation of cells with growth factors. For this reason, D-cyclins are regarded as links between the cellular environment and the cell cycle machinery (6).
Cell cycle inhibitors play an important role in regulating the activity of cyclin D–CDK4/6 (Fig. 1). The INK inhibitors (p16INK4A, p15INK4B, p18INK4C, p19INK4D) bind to CDK4 or CDK6 and prevent their interaction with D-type cyclins, thereby inhibiting cyclin D–CDK4/6 kinase activity. By contrast, KIP/CIP inhibitors (p27KIP1, p57KIP2, p21CIP1), which inhibit the activity of CDK2-containing complexes, serve as assembly factors for cyclin D–CDK4/6 (69). This was demonstrated by the observation that mouse fibroblasts devoid of p27KIP1 and p21CIP1 fail to assemble cyclin D–CDK4/6 complexes (10).
Fig. 1. Molecular events governing progression through the G1 phase of the cell cycle.
The mammalian cell cycle can be divided into G1, S (DNA synthesis), G2, and M (mitosis) phases. During G1 phase, cyclin D (CycD)–CDK4/6 kinases together with cyclin E (CycE)–CDK2 phosphorylate the retinoblastoma protein RB1. This activates the E2F transcriptional program and allows entry of cells into S phase. Members of the INK family of inhibitors (p16INK4A, p15INK4B, p18INK4C, and p19INK4D) inhibit cyclin D–CDK4/6; KIP/CIP proteins (p21CIP1, p27KIP1, and p57KIP2) inhibit cyclin E–CDK2. Cyclin D–CDK4/6 complexes use p27KIP1 and p21CIP1 as “assembly factors” and sequester them away from cyclin E–CDK2, thereby activating CDK2. Proteins that are frequently lost or down-regulated in cancers are marked with green arrows, overexpressed proteins with red arrows.
p27KIP1 can bind cyclin D–CDK4/6 in an inhibitory or noninhibitory mode, depending on p27KIP1 phosphorylation status. Cyclin D–p27KIP1-CDK4/6 complexes are catalytically inactive unless p27KIP1 is phosphorylated on Tyr88 and Tyr89 (11). Two molecular mechanisms may explain this switch. First, Tyr88/Tyr89 phosphorylation may dislodge the helix of p27KIP1 from the CDK active site and allow adenosine triphosphate (ATP) binding (12). Second, the presence of tyrosine-unphosphorylated p27KIP1 within the cyclin D–CDK4 complex prevents the activating phosphorylation of CDK4’s T-loop by the CDK-activating kinase (CAK) (12). Brk has been identified as a physiological kinase of p27KIP1 (13); Abl and Lyn can phosphorylate p27KIP1 in vitro, but their in vivo importance remains unclear (1114).
The activity of cyclin D–CDK4/6 is also regulated by proteolysis. Cyclin D1 is an unstable protein with a half-life of less than 30 min. At the end of G1 phase, cyclin D1 is phosphorylated at Thr286 by GSK3β (15). This facilitates association of cyclin D1 with the nuclear exportin CRM1 and promotes export of cyclin D1 from the nucleus to the cytoplasm (16). Subsequently, phosphorylated cyclin D1 becomes polyubiquitinated by E3 ubiquitin ligases, thereby targeting it for proteasomal degradation. Several substrate receptors of E3 ubiquitin ligases have been implicated in recognizing phosphorylated cyclin D1, including F-box proteins FBXO4 (along with αB crystallin), FBXO31, FBXW8, β-TrCP1/2, and SKP2 (17). The anaphase-promoting complex/cyclosome (APC/C) was also proposed to target cyclin D1 while F-box proteins FBXL2 and FBXL8 target cyclins D2 and D3 (1718). Surprisingly, the level and stability of cyclin D1 was unaffected by depletion of several of these proteins, indicating that some other E3 plays a rate-limiting role in cyclin D1 degradation (19). Indeed, recent studies reported that D-cyclins are ubiquitinated and targeted for proteasomal degradation by the E3 ubiquitin ligase CRL4, which uses AMBRA1 protein as its substrate receptor (2022).

Cyclin D–CDK4/6 in cancer

Genomic aberrations of the cyclin D1 gene (CCND1) represent frequent events in different tumor types. The t(11;14)(q13;q32) translocation juxtaposing CCND1 with the immunoglobulin heavy-chain (IGH) locus represents the characteristic feature of mantle-cell lymphoma and is frequently observed in multiple myeloma or plasma cell leukemia (2324). Amplification of CCND1 is seen in many other malignancies—for example, in 13 to 20% of breast cancers (2324), more than 40% of head and neck squamous cell carcinomas, and more than 30% of esophageal squamous cell carcinomas (23). A higher proportion of cancers (e.g., up to 50% of mammary carcinomas) overexpress cyclin D1 protein (24). Also, cyclins D2 and D3, CDK4, and CDK6 are overexpressed in various tumor types (59). Cyclin D–CDK4/6 can also be hyperactivated through other mechanisms such as deletion or inactivation of INK inhibitors, most frequently p16INK4A (5923). Altogether, a very large number of human tumors contain lesions that hyperactivate cyclin D–CDK4/6 (5).
An oncogenic role for cyclin D–CDK4/6 has been supported by mouse cancer models. For example, targeted overexpression of cyclin D1 in mammary glands of transgenic mice led to the development of mammary carcinomas (25). Also, overexpression of cyclin D2, D3, or CDK4, or loss of p16INK4a resulted in tumor formation (9).
Conversely, genetic ablation of D-cyclins, CDK4, or CDK6 decreased tumor sensitivity (9). For instance, Ccnd1– or Cdk4-null mice, or knock-in mice expressing kinase-inactive cyclin D1–CDK4/6, were resistant to develop human epidermal growth factor receptor 2 (HER2)–driven mammary carcinomas (2629). An acute, global shutdown of cyclin D1 in mice bearing HER2-driven tumors arrested tumor growth and triggered tumor-specific senescence while having no obvious impact on normal tissues (30). Likewise, an acute ablation of CDK4 arrested tumor cell proliferation and triggered tumor cell senescence in a KRAS-driven non–small-cell lung cancer (NSCLC) mouse model (31). These observations indicated that CDK4 and CDK6 might represent excellent therapeutic targets in cancer treatment.

CDK4/6 functions in cell proliferation and oncogenesis

The best-documented function of cyclin D–CDK4/6 in driving cell proliferation is phosphorylation of the retinoblastoma protein, RB1, and RB-like proteins, RBL1 and RBL2 (56) (Fig. 1). Unphosphorylated RB1 binds and inactivates or represses E2F transcription factors. According to the prevailing model, phosphorylation of RB1 by cyclin D–CDK4/6 partially inactivates RB1, leading to release of E2Fs and up-regulation of E2F-transcriptional targets, including cyclin E. Cyclin E forms a complex with its kinase partner, CDK2, and completes full RB1 phosphorylation, leading to activation of the E2F transcriptional program and facilitating S-phase entry (56). In normal, nontransformed cells, the activity of cyclin D–CDK4/6 is tightly regulated by the extracellular mitogenic milieu. This links inactivation of RB1 with mitogenic signals. In cancer cells carrying activating lesions in cyclin D–CDK4/6, the kinase is constitutively active, thereby decoupling cell division from proliferative and inhibitory signals (5).
This model has been questioned by the demonstration that RB1 exists in a monophosphorylated state throughout G1 phase and becomes inactivated in late G1 by cyclin E–CDK2, which “hyperphosphorylates” RB1 on multiple residues (32). However, recent single-cell analyses revealed that cyclin D–CDK4/6 activity is required for the hyperphosphorylation of RB1 throughout G1, whereas cyclin E/A–CDK maintains RB1 hyperphosphorylation in S phase (33). Moreover, phosphorylation of RB1 by cyclin D–CDK4/6 was shown to be required for normal cell cycle progression (34).
In addition to this kinase-dependent mechanism, up-regulation of D-cyclin expression and formation of cyclin D–CDK4/6 complexes lead to redistribution of KIP/CIP inhibitors from cyclin E–CDK2 complexes (which are inhibited by these proteins) to cyclin D–CDK4/6 (which use them as assembly factors), thereby activating the kinase activity of cyclin E–CDK2 (6). Cyclin E–CDK2 in turn phosphorylates RB1 and other cellular proteins and promotes cell cycle progression.
Cyclin D1–CDK4/6 directly phosphorylates, stabilizes, and activates the transcription factor FOXM1. This promotes cell cycle progression and protects cancer cells from entering senescence (35). Cyclin D–CDK4 also phosphorylates and inactivates SMAD3, which mediates transforming growth factor–β (TGF-β) antiproliferative response. CDK4/6-dependent phosphorylation of SMAD3 inhibits its transcriptional activity and disables the ability of TGF-β to induce cell cycle arrest (36). FZR1/CDH1, an adaptor protein of the APC complex, is another phosphorylation substrate of CDK4. Depletion of CDH1 in human cancer cells partially rescued the proliferative block upon CDK4/6 inhibition, and it cooperated with RB1 depletion in restoring full proliferation (37).
Cyclin D–CDK4/6 also phosphorylates and inactivates TSC2, a negative regulator of mTORC1, thereby resulting in mTORC1 activation. Conversely, inhibition of CDK4/6 led to decreased mTORC1 activity and reduced protein synthesis in cells representing different human tumor types. It was proposed that through TSC2 phosphorylation, activation of cyclin D–CDK4/6 couples cell growth with cell division (38). Consistent with this, the antiproliferative effect of CDK4/6 inhibition was reduced in cells lacking TSC2 (38).
MEP50, a co-regulatory factor of protein arginine-methyltransferase 5 (PRMT5), is phosphorylated by cyclin D1–CDK4. Through this mechanism, cyclin D1–CDK4/6 increases the catalytic activity of PRMT5/MEP50 (39). It was proposed that deregulation of cyclin D1–CDK4 kinase in tumor cells, by increasing PRMT5/MEP50 activity, reduces the expression of CUL4, a component of the E3 ubiquitin-ligase complex, and stabilizes CUL4 targets such as CDT1 (39). In addition, by stimulating PRMT5/MEP50-dependent arginine methylation of p53, cyclin D–CDK4/6 suppresses the expression of key antiproliferative and pro-apoptotic p53 target genes (40). Another study proposed that PRMT5 regulates splicing of the transcript encoding MDM4, a negative regulator of p53. CDK4/6 inhibition reduced PRMT5 activity and altered the pre-mRNA splicing of MDM4, leading to decreased levels of MDM4 protein and resulting in p53 activation. This, in turn, up-regulated the expression of a p53 target, p21CIP1, that blocks cell cycle progression (41).
During oncogenic transformation of hematopoietic cells, chromatin-bound CDK6 phosphorylates the transcription factors NFY and SP1 and induces the expression of p53 antagonists such as PRMT5, PPM1D, and MDM4 (42). Also, in acute myeloid leukemia cells expressing constitutively activated FLT3, CDK6 binds the promoter region of the FLT3 gene as well as the promoter of PIM1 pro-oncogenic kinase and stimulates their expression. Treatment of FLT3-mutant leukemic cells with a CDK4/6 inhibitor decreased FLT3 and PIM1 expression and triggered cell cycle arrest and apoptosis (43). The relevance of these various mechanisms in the context of human tumors is unclear and requires further study.

Mechanism of action of CDK4/6 inhibitors

Three small-molecule CDK4/6 inhibitors have been extensively characterized in preclinical studies: palbociclib and ribociclib, which are highly specific CDK4/6 inhibitors, and abemaciclib, which inhibits CDK4/6 and other kinases (Table 1). It has been assumed that these compounds act in vivo by directly inhibiting cyclin D–CDK4/6 (9). This simple model has been recently questioned by observations that palbociclib inhibits only cyclin D–CDK4/6 dimers, but not trimeric cyclin D–CDK4/6-p27KIP1 (44). However, it is unlikely that substantial amounts of cyclin D–CDK4 dimers ever exist in cells, because nearly all cyclin D–CDK4 in vivo is thought to be complexed with KIP/CIP proteins (111444). Palbociclib also binds monomeric CDK4 (44). Surprisingly, treatment of cancer cells with palbociclib for 48 hours failed to inhibit CDK4 kinase, despite cell cycle arrest, but it inhibited CDK2 (44). Hence, palbociclib might prevent the formation of active CDK4-containing complexes (through binding to CDK4) and indirectly inhibit CDK2 by liberating KIP/CIP inhibitors. This model needs to be reconciled with several observations. First, treatment of cells with CDK4/6 inhibitors results in a rapid decrease of RB1 phosphorylation on cyclin D–CDK4/6-dependent sites, indicating an acute inhibition of CDK4/6 (4547). Moreover, CDK4/6 immunoprecipitated from cells can be inhibited by palbociclib (48) and p21CIP-associated cyclin CDK4/6 kinase is also inhibited by treatment of cells with palbociclib (49). Lastly, CDK2 is dispensable for proliferation of several cancer cell lines (5051), hence the indirect inhibition of CDK2 alone is unlikely to be responsible for cell cycle arrest.
Name of compound IC50 Other known targets Stage of clinical development
Palbociclib (PD-0332991) D1-CDK4, 11 nM;
D2-CDK6, 15 nM;
D3-CDK4, 9 nM
FDA-approved for HR+/HER2 advanced
breast cancer in combination with
endocrine therapy; phase 2/3 trials
for several other tumor types
Ribociclib (LEE011) D1-CDK4, 10 nM;
D3-CDK6, 39 nM
FDA-approved for HR+/HER2 advanced
breast cancer in combination with
endocrine therapy; phase 2/3 trials
for several other tumor types
Abemaciclib (LY2835219) D1-CDK4, 0.6 to 2 nM;
D3-CDK6, 8 nM
Cyclin T1–CDK9, PIM1, HIPK2, CDKL5,
GSK3α/β, and (at higher doses)
cyclin E/A–CDK2 and cyclin B–CDK1
FDA-approved for early (adjuvant) and
advanced HR+/HER2 breast cancer in
combination with endocrine therapy;
FDA-approved as monotherapy in advanced
HR+/HER2 breast cancer; phase 2/3 trials
for several other tumor types
Trilaciclib (G1T28) D1-CDK4, 1 nM;
D3-CDK6, 4 nM
FDA-approved for small-cell lung cancer
to reduce chemotherapy-induced bone
marrow suppression; phase 2/3 trials
for other solid tumors
Lerociclib (G1T38) D1-CDK4, 1 nM;
D3-CDK6, 2 nM
Phase 1/2 trials for HR+/HER2 advanced
breast cancer and EGFR-mutant
non–small-cell lung cancer
SHR6390 CDK4, 12 nM;
CDK6, 10 nM
Phase 1/2/3 trials for HR+/HER2 advanced
breast cancer and other solid tumors
PF-06873600 CDK4, 0.13 nM (Ki),
CDK6, 0.16 nM (Ki)
CDK2, 0.09 nM (Ki) Phase 2 trials for HR+/HER2 advanced
breast cancer and other solid tumors
FCN-437 D1-CDK4, 3.3 nM;
D3-CDK6, 13.7 nM
Phase 1/2 trials for HR+/HER2 advanced
breast cancer and other solid tumors
Birociclib (XZP-3287) Not reported Phase 1/2 trials for HR+/HER2 advanced
breast cancer and other solid tumors
HS-10342 Not reported Phase 1/2 trials for HR+/HER2 advanced
breast cancer and other solid tumors
CS3002 Not reported Phase 1 trial for solid tumors

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Table 1. Currently available CDK4/6 inhibitors.
This table lists major inhibitors of CDK4 and CDK6, half-maximal inhibitory concentration (IC50) for different cyclin D–CDK4/6 complexes (if known), other known targets, and the stage of clinical development. Ki, inhibitory constant.
Palbociclib, ribociclib, and abemaciclib were shown to block binding of CDK4 and CDK6 to CDC37, the kinase-targeting subunit of HSP90, thereby preventing access of CDK4/6 to the HSP90-chaperone system (52). Because the HSP90-CDC37 complex stabilizes several kinases (53), these observations suggest that CDK4/6 inhibitors, by disrupting the interaction between CDC37 and CDK4 or CDK6, might promote degradation of CDK4 and CDK6. However, depletion of CDK4/6 is typically not observed upon treatment with CDK4/6 inhibitors (54). More studies are needed to resolve these conflicting reports and to establish how CDK4/6 inhibitors affect the cell cycle machinery in cancer cells.

Validation of CDK4/6 inhibitors as anticancer agents

Consistent with the notion that RB1 represents the major rate-limiting substrate of cyclin D–CDK4/6 in cell cycle progression (5557), palbociclib, ribociclib, and abemaciclib were shown to block proliferation of several RB1-positive cancer cell lines, but not cell lines that have lost RB1 expression (465859). Breast cancer cell lines representing the luminal, estrogen receptor–positive (ER+) subtype were shown to be most susceptible to cell proliferation arrest upon palbociclib treatment (45). Palbociclib, ribociclib, abemaciclib, and another CDK4/6 inhibitor, lerociclib, were demonstrated to display potent antitumor activity in xenografts of several tumor types, including breast cancers (466062). Palbociclib and abemaciclib cross the blood-brain barrier and inhibit growth of intracranial glioblastoma (GBM) xenografts, with abemaciclib being more efficient in reaching the brain (6364). Recently, additional CDK4/6 inhibitors were shown to exert therapeutic effects in mouse xenograft models of various cancer types, including SHR6390 (65), FCN-437 (66), and compound 11 (67); the latter two were reported to cross the blood-brain barrier. In most in vivo studies, the therapeutic effect was dependent on expression of intact RB1 protein in tumor cells (4663). However, antitumor effects of palbociclib were also reported in bladder cancer xenografts independently of RB1 status; this was attributed to decreased phosphorylation of FOXM1 (68).

Tumor cell senescence upon CDK4/6 inhibition

In addition to blocking cell proliferation, inhibition of CDK4/6 can also trigger tumor cell senescence (63), which depends on RB1 and FOXM1 (3554). The role of RB1 in enforcing cellular senescence is well established (69). In addition, cyclin D–CDK4/6 phosphorylates and activates FOXM1, which has anti-senescence activity (3570). Senescence represents a preferred therapeutic outcome to cell cycle arrest, as it may lead to a durable inhibition of tumor growth.
It is not clear what determines the extent of senescence upon treatment of cancer cells with CDK4/6 inhibitors. A recent study showed that inhibition of CDK4/6 leads to an RB1-dependent increase in reactive oxygen species (ROS) levels, resulting in activation of autophagy, which mitigates the senescence of breast cancer cells in vitro and in vivo (71). Co-treatment with palbociclib plus autophagy inhibitors strongly augmented the ability of CDK4/6 inhibitors to induce tumor cell senescence and led to sustained inhibition of cancer cell proliferation in vitro and of xenograft growth in vivo (71). Decreased mTOR signaling after long-term CDK4/6 inhibition was shown to be essential for the induction of senescence in melanoma cells, and activation of mTORC1 overrode palbociclib-induced senescence (72). Others postulated that expression of the chromatin-remodeling enzyme ATRX and degradation of MDM2 determines the choice between quiescence and senescence upon CDK4/6 inhibition (73). Inhibition of CDK4 causes dissociation of the deubiquitinase HAUSP/USP7 from MDM2, thereby driving autoubiquitination and proteolytic degradation of MDM2, which in turn promotes senescence. This mechanism requires ATRX, which suggests that expression of ATRX can be used to predict the senescence response (73). Two additional proteins that play a role in this process are PDLIM7 and type II cadherin CDH18. Expression of CDH18 correlated with a sustained response to palbociclib in a phase 2 trial for patients with liposarcoma (74).

Markers predicting response to CDK4/6 inhibition

Only tumors with intact RB1 respond to CDK4/6 inhibitor treatment by undergoing cell cycle arrest or senescence (958). In addition, “D-cyclin activating features” (CCND1 translocation, CCND2 or CCND3 amplification, loss of the CCND1-3 3′-untranslated region, and deletion of FBXO31 encoding an F-box protein implicated in cyclin D1 degradation) were shown to confer a strong response to abemaciclib in cancer cell lines (58). Moreover, co-deletion of CDKN2A and CDKN2C (encoding p16INK4A/p19ARF and p18INK4C, respectively) confers palbociclib sensitivity in glioblastoma (75). Thr172 phosphorylation of CDK4 and Tyr88 phosphorylation of p27KIP1 (both associated with active cyclin D–CDK4) correlate with sensitivity of breast cancer cell lines or tumor explants to palbociclib (7677). Surprisingly, in PALOMA-1, PALOMA-2, and PALOMA-3 trials (7880), and in another independent large-scale study (81), CCND1 gene amplification or elevated levels of cyclin D1 mRNA or protein were not predictive of palbociclib efficacy. Conversely, overexpression of CDK4, CDK6, or cyclin E1 is associated with resistance of tumors to CDK4/6 inhibitors (see below).

Synergy of CDK4/6 inhibitors with other compounds

Several preclinical studies have documented the additive or synergistic effects of combining CDK4/6 inhibitors with inhibitors of the receptor tyrosine kinases as well as phosphoinositide 3-kinase (PI3K), RAF, or MEK (Table 2). This synergism might be because these pathways impinge on the cell cycle machinery through cyclin D–CDK4/6 (8286). In some cases, the effect was seen in the presence of specific genetic lesions, such as EGFRBRAFV600EKRAS, and PIK3CA mutations (598789) (Table 2). When comparing different dosing regimens, continuous treatment with a MEK inhibitor with intermittent palbociclib resulted in more complete tumor responses than other combination schedules (90). Treatment with CDK4/6 inhibitors sensitized cancer cells to ionizing radiation (63) or cisplatin (68). The synergism with platinum-based chemotherapy was attributed to the observation that upon this treatment, CDK6 phosphorylates and stabilizes the FOXO3 transcription factor, thereby promoting tumor cell survival. Consequently, inhibition of CDK6 increases platinum sensitivity by enhancing tumor cell death (91).
CDK4/6 inhibitor Synergistic target Inhibitor Disease
Palbociclib PI3K Taselisib, pictilisib PIK3CA mutant TNBC
AR Enzalutamide Androgen receptor–positive TNBC
EGFR Erlotinib TNBC, esophageal squamous cell carcinoma
RAF PLX4720 BRAF-V600E mutant melanoma
MEK Trametinib KRAS mutant colorectal cancer
MEK PD0325901 (mirdametinib) KRAS or BRAFV600E mutant colorectal cancer
MEK MEK162 (binimetinib) KRAS mutant colorectal cancer
MEK AZD6244 (selumetinib) Pancreatic ductal adenocarcinoma
PI3K/mTOR BEZ235 (dactolisib), AZD0855, GDC0980 (apitolisib) Pancreatic ductal adenocarcinoma
IGF1R/InsR BMS-754807 Pancreatic ductal adenocarcinoma
mTOR Temsirolimus Pancreatic ductal adenocarcinoma
mTOR AZD2014 (vistusertib) ER+ breast cancer
mTOR MLN0128 (sapanisertib) Intrahepatic cholangiocarcinoma
mTOR Everolimus Melanoma, glioblastoma
Ribociclib PI3K GDC-0941 (pictilisib), BYL719 (alpelisib) PIK3CA mutant breast cancer
PDK1 GSK2334470 ER+ breast cancer
EGFR Nazartinib EGFR-mutant lung cancer
RAF Encorafenib BRAF-V600E mutant melanoma
mTOR Everolimus T-ALL
Inflammation Glucocorticoid dexamethasone T-ALL
γ-Secretase Compound E T-ALL
Abemaciclib HER2 Trastuzumab HER2+ breast cancer
EGFR and HER2 Lapatinib HER2+ breast cancer
RAF LY3009120, vemurafenib KRAS mutant lung or colorectal cancer, NRAS or
BRAF-V600E mutant melanoma
Temozolomide (alkylating agent) Glioblastoma

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Table 2. Combination treatments that demonstrated synergy with CDK4/6 inhibitors in preclinical studies.
TNBC, triple-negative breast cancer; AR, androgen receptor; ER+, estrogen receptor–positive; T-ALL, T cell acute lymphoblastic leukemia; HER2+, human epidermal growth factor receptor 2–positive; PI3K, phosphoinositide 3-kinase; EGFR, epidermal growth factor receptor; IGF1R, insulin-like growth factor 1 receptor, InsR, insulin receptor.
In several instances, co-treatment with CDK4/6 inhibitors prevented the development of resistance to other compounds or inhibited the proliferation of resistant tumor cells. Co-treatment of melanoma patient-derived xenografts (PDXs) with ribociclib plus the RAF inhibitor encorafenib delayed or prevented development of encorafenib resistance (92). PDXs that acquired encorafenib resistance remained sensitive to the combination of encorafenib plus ribociclib (59). Treatment of BRAFV600E-mutant melanoma xenografts with palbociclib plus the BRAFV600E inhibitor PLX4720 prevented development of resistance (89). BRAFV600E-mutant melanoma cell lines that acquired resistance to the BRAFV600E inhibitor vemurafenib remained sensitive to palbociclib or abemaciclib, and xenografts underwent senescence and tumor regression upon CDK4/6 inhibition (7293). Treatment of ALK-mutant, ALK kinase inhibitor–resistant neuroblastoma xenografts with palbociclib restored the sensitivity to these compounds (94). A combination of PI3K and CDK4/6 inhibitors overcame the intrinsic and acquired resistance of breast cancers to PI3K inhibitors and resulted in regression of PIK3CA-mutant xenografts (88).
Up-regulation of cyclin D1 expression was shown to mediate acquired resistance of HER2+ tumors to anti-HER2 therapies in a mouse breast cancer model (95). Treatment of mice bearing trastuzumab-resistant tumors or PDXs of resistant HER2+ mammary carcinomas with abemaciclib restored the sensitivity of tumors to HER2 inhibitors and inhibited tumor cell proliferation. Moreover, in the case of treatment-naïve tumors, co-administration of abemaciclib significantly delayed the development of resistance to anti-HER2 therapies (95).
Several anticancer treatments, such as chemotherapy, target dividing cells. Because CDK4/6 inhibitors block tumor cell proliferation, they might impede the effects of chemotherapy. Indeed, several reports have documented that co-administration of CDK4/6 inhibitors antagonized the antitumor effects of compounds that act during S phase (doxorubicin, gemcitabine, methotrexate, mercaptopurine) or mitosis (taxanes) (9697). However, some authors reported synergistic effects (9899), although the molecular underpinnings are unclear.
A recent report documented that administration of CDK4/6 inhibitors prior to taxanes inhibited tumor cell proliferation and impeded the effect of taxanes (100). By contrast, administration of taxanes first (or other chemotherapeutic compounds that act on mitotic cells or cells undergoing DNA synthesis), followed by CDK4/6 inhibitors, had a strong synergistic effect. The authors showed that by repressing the E2F-dependent transcriptional program, CDK4/6 inhibitors impaired the expression of genes required for DNA-damage repair via homologous recombination. Because treatment of cancer cells with chemotherapy triggers DNA damage, the impairment of DNA-damage repair induced cytotoxicity, thereby explaining the synergistic effect (100).
Cells with impaired homologous recombination rely on poly-(ADP-ribose) polymerase (PARP) for double-stranded DNA-damage repair, which renders them sensitive to PARP inhibition. Indeed, a strong synergistic effect has been demonstrated between CDK4/6 inhibitors and PARP inhibitors in PDX-derived cell lines (100). Such synergy was also reported for ovarian cancer cells (101). Another study found that inhibition of CDK4/6 resulted in down-regulation of PARP levels (102).

Protection against chemotherapy-induced toxicity

Administration of palbociclib to mice induced reversible quiescence in hematopoietic stem/progenitor cells (HSPCs). This effect protected mice from myelosuppression after total-body irradiation. Moreover, treatment of tumor-bearing mice with CDK4/6 inhibitors together with irradiation mitigated radiation-induced toxicity without compromising the therapeutic effect (103). Co-administration of a CDK4/6 inhibitor, trilaciclib, with cytotoxic chemotherapy (5-FU, etoposide) protected animals from chemotherapy-induced exhaustion of HSPCs, myelosuppression, and apoptosis of bone marrow (60104). These observations led to phase 2 clinical trial, which evaluated the effects of trilaciclib administered prior to etoposide and carboplatin for treatment of small-cell lung cancer. Trilaciclib improved myelopreservation while having no adverse effect on antitumor efficacy (105). A similar phase 2 clinical trial investigating trilaciclib in combination with gemcitabine and carboplatin chemotherapy in patients with metastatic triple-negative breast cancer (TNBC) did not observe a significant difference in myelosuppression. However, this study demonstrated an overall survival benefit of the combination therapy (106107).

Metabolic function of CDK4/6 in cancer cells

The role of CDK4/6 in tumor metabolism is only starting to be appreciated (Fig. 2A). Treatment of pancreatic cancer cells with CDK4/6 inhibitors was shown to induce tumor cell metabolic reprogramming (108). CDK4/6 inhibition increased the numbers of mitochondria and lysosomes, activated mTOR, and increased the rate of oxidative phosphorylation, likely through an RB1-dependent mechanism (108). Combined inhibition of CDK4/6 and mTOR strongly suppressed tumor cell proliferation (108). Moreover, CDK4/6 can phosphorylate and inactivate TFEB, the master regulator of lysosomogenesis, and through this mechanism reduce lysosomal numbers. Conversely, CDK4/6 inhibition activated TFEB and increased the number of lysosomes (109). Another mechanism linking CDK4/6 and lysosomes was provided by the observation that treatment of TNBC cells with CDK4/6 inhibitors decreased mTORC1 activity and impaired the recruitment of mTORC1 to lysosomes (110). Consistent with the idea that mTORC1 inhibits lysosomal biogenesis, CDK4/6 inhibition increased the number of lysosomes in tumor cells. Because an increased lysosomal biomass underlies some cases of CDK4/6 inhibitor resistance (see below) (111), stimulation of lysosomogenesis by CDK4/6 inhibitors might limit their clinical efficacy by inducing resistance.
Fig. 2. CDK4 and CDK6: More than cell cycle kinases.
Although the role of CDK4 and CDK6 in cell cycle progression has been well documented, both kinases regulate several other functions that are only now starting to be unraveled. (A) Inhibition of CDK4/6 (CDK4/6i) affects lysosome and mitochondrial numbers as well as oxidative phosphorylation. Cyclin D3–CDK6 phosphorylates glycolytic enzymes 6-phosphofructokinase (PFKP) and pyruvate kinase M2 (PKM2), thereby controlling ROS levels via the pentose phosphate (PPP) and serine synthesis pathways. (B) Inhibition of CDK4/6 affects antitumor immunity, acting both within cancer cells and on the immune system of the host. In tumor cells, inhibition of CDK4/6 impedes expression of an E2F target, DNA methyltransferase (DNMT). DNMT inhibition reduces methylation of endogenous retroviral genes (ERV) and increases intracellular levels of double-stranded RNA (dsRNA) (114). In effector T cells, inhibition of CDK4/6 stimulates NFAT transcriptional activity and enhances secretion of IFN-γ and interleukin 2 (IL-2) (115).
Lastly, CDK4/6 inhibition impaired lysosomal function and the autophagic flux in cancer cells. It was argued that this lysosomal dysfunction was responsible for the senescent phenotype in CDK4/6 inhibitor–treated cells (110). Because lysosomes are essential for autophagy, the authors co-treated TNBC xenografts with abemaciclib plus an AMPK activator, A769662 (which induces autophagy), and found that this led to cancer cell death and subsequent regression of tumors (110).
Cyclin D3–CDK6 phosphorylates and inhibits two rate-limiting glycolytic enzymes, 6-phosphofructokinase and pyruvate kinase M2. This redirects glycolytic intermediates into the pentose phosphate pathway (PPP) and serine synthesis pathway. Through this mechanism, cyclin D3–CDK6 promotes the production of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and reduced glutathione (GSH) and helps to neutralize ROS (112). Treatment of tumors expressing high levels of cyclin D3–CDK6 (such as leukemias) with CDK4/6 inhibitors reduced the PPP- and serine-synthesis pathway flow, thereby depleting the antioxidants NADPH and GSH. This increased ROS levels and triggered tumor cell apoptosis (112).
Another link between cyclin D–CDK4/6 in metabolism and cancer was provided by the observation that livers of obese/diabetic mice up-regulate cyclin D1 expression (113). Treatment of these mice with an antidiabetic compound, metformin, reduced liver cyclin D1 levels and largely protected mice against development of hepatocellular carcinoma. Also, genetic ablation of cyclin D1 protected obese/diabetic mice from liver cancer, and administration of palbociclib inhibited liver cancer progression. These treatments had no effect on tumors in nonobese animals (113). These observations raise the possibility of using antidiabetic compounds with CDK4/6 inhibitors for treatment of liver cancers in obese patients.

CDK4/6 inhibitors and antitumor immune responses

Several recent reports have started to unravel how inhibition of CDK4/6 influences antitumor immune responses, acting both on tumor cells as well as on the tumor immune environment (Fig. 2B). Treatment of breast cancer–bearing mice or breast cancer cells with abemaciclib activated expression of endogenous retroviral elements in tumor cells, thereby increasing the levels of double-stranded RNA. This, in turn, stimulated production of type III interferons and increased presentation of tumor antigens. Hence, CDK4/6 inhibitors, by inducing viral gene expression, trigger antiviral immune responses that help to eliminate the tumor (114).
Inhibition of CDK4/6 also affects the immune system by impeding the proliferation of CD4+FOXP3+ regulatory T cells (Tregs), which normally inhibit the antitumor response. Because cytotoxic CD8+ T cells are less affected by CDK4/6 inhibition, abemaciclib treatment decreases the Treg/CD8+ ratio of intratumoral T cells and facilitates tumor cell killing by cytotoxic CD8+ T cells (114).
Inhibition of CDK4/6 also resulted in activation of T cells through derepression of NFAT signaling. NFAT4 (and possibly other NFATs) are phosphorylated by cyclin D3–CDK6 (115). Inhibition of CDK4/6 decreased phosphorylation of NFATs, resulting in their nuclear translocation and enhanced transcriptional activity. This caused up-regulation of NFAT targets, resulting in T cell activation, which enhanced the antitumor immune response. In addition, CDK4/6 inhibitors increased the infiltration of effector T cells into tumors, likely because of elevated levels of chemokines CXCL9 and CXCL10 after CDK4/6 inhibitor treatment (115). Abemaciclib treatment also induced inflammatory and activated T cell phenotypes in tumors and up-regulated the expression of immune checkpoint proteins CD137, PD-L1, and TIM-3 on CD4+ and CD8+ cells (116).
CDK4/6 inhibition also caused up-regulation of PD-L1 protein expression in tumor cells (117118). This effect was shown to be independent of RB1 status in the tumor. Mechanistically, CDK4/6 phosphorylates and stabilizes SPOP, which promotes PD-L1 polyubiquitination and degradation (118). Cyclin D–CDK4 also represses expression of PD-L1 through RB1. Specifically, cyclin D–CDK4/6-mediated phosphorylation of RB1 on S249/T252 promotes binding of RB1 to NF-κB protein p65, and this represses the expression of a subset NF-κB–regulated genes, including PD-L1 (119).
These observations prompted tests of the efficacy of combining CDK4/6 inhibitors with antibodies that elicit immune checkpoint blockade. Indeed, treatment of mice bearing autochthonous breast cancers, or cancer allografts, with CDK4/6 inhibitors together with anti-PD-1/PD-L1 antibodies enhanced the efficacy of immune checkpoint blockade and led to complete tumor regression in a high proportion of animals (114115118). Conversely, activation of the cyclin D–CDK4 pathway by genomic lesions in human melanomas correlated with resistance to anti–PD-1 therapy (117).
Some authors did not observe synergy when abemaciclib was administered concurrently with immune checkpoint inhibitors in allograft tumor models (116120). However, a strong synergistic antitumor effect was detected when abemaciclib was administered first (and continued) and anti–PD-L1 antibody was administered later. The combined treatment induced immunological memory, as mice that underwent tumor regression were resistant to rechallenge with the same tumor (116). Abemaciclib plus anti–PD-L1 treatment increased infiltration of CD4+ and CD8+ T cells into tumors, and increased the expression of major histocompatibility complex class I (MHC-I) and MHC-II on tumor cells and on macrophages and MHC-I on dendritic cells (116). In the case of anti–CTLA-4 plus anti–PD-1 treatment in melanoma allograft model, the synergistic effect was observed when immune checkpoint inhibitor treatment was started first, followed by abemaciclib (120).
The synergistic antitumor effect of PI3K and CDK4/6 inhibitors in TNBC is mediated, in part, by enhancement of tumor immunogenicity (121). Combined treatment of TNBC cells with ribociclib plus the PI3K inhibitor apelisib synergistically up-regulated the expression of immune-related pathways in tumor cells, including proteins involved in antigen presentation. Co-treatment of tumor-bearing mice also decreased proliferation of CD4+FOXP3+ Treg cells, increased activation of intratumoral CD4+ and CD8+ T cells, increased the frequency of tumor-infiltrating NKT cells, and decreased the numbers of intratumoral immunosuppressive myeloid-derived suppressor cells. Moreover, combined treatment strongly augmented the response to immune checkpoint therapy with PD-1 and CTLA-4 antibodies (121).
Single-cell RNA sequencing of human melanomas identified an immune resistance program expressed by tumor cells that correlates with T cell exclusion from the tumor mass and immune evasion by tumor cells. The program can predict the response of tumors to immune checkpoint inhibitors. Treatment of human melanoma cells with abemaciclib repressed this program in an RB1-dependent fashion (120).
Together, these findings indicate that CDK4/6 inhibitors may convert immunologically “cold” tumors into “hot” ones. The most pressing issue is to validate these findings in a clinical setting. The utility of combining CDK4/6 inhibitors with PD-1 or PD-L1 antibodies is currently being evaluated in several clinical trials. Note that the effects of CDK4/6 inhibition on the immune system of the host are independent of tumor cell RB1 status, raising the possibility of using CDK4/6 inhibitors to also boost the immune response against RB1-negative tumors.

CDK4/6 inhibitors in clinical trials

Table 3 summarizes major clinical trials with CDK4/6 inhibitors. Given early preclinical data indicating that breast cancers—in particular, the hormone receptor–positive ones—are very sensitive to CDK4/6 inhibition (as discussed above), many clinical trials have focused on this cancer type. Most studies have evaluated CDK4/6 inhibitors administered together with anti-estrogens (the aromatase inhibitors letrozole or anastrozole, or the estrogen receptor antagonist fulvestrant) for treatment of advanced/metastatic HR+/HER2 breast cancers in postmenopausal women. Addition of CDK4/6 inhibitors significantly extended median progression-free survival (78122130) and prolonged median overall survival (131134). Moreover, abemaciclib has shown clinical activity when administered as a single agent (135). Consequently, palbociclib, ribociclib, and abemaciclib have been approved by the US Food and Drug Administration (FDA) for treatment of patients with advanced/metastatic HR+/HER2 breast cancer (Box 1). A recent phase 3 clinical trial, MonarchE, evaluated abemaciclib plus standard endocrine therapy in treatment of patients with early-stage, high-risk, lymph node–positive HR+/HER2 breast cancer. Addition of abemaciclib reduced the risk of breast cancer recurrence (136). This is in contrast to the similar PALLAS study reported this year, which found no benefit of adding palbociclib to endocrine therapy for women with early-stage breast cancer (137). Analysis of patient populations in these two trials may help to explain the different outcomes. It is also possible that the favorable outcome of the MonarchE study reflects a broader spectrum of kinases inhibited by abemaciclib. The utility of CDK4/6 inhibitors in early-stage breast cancer remains unclear and is being addressed in ongoing clinical trials (PALLAS, PENELOPE-B, EarLEE-1, MonarchE) (138).
Trial name Trial details Treatment Patients Outcome Ref. Other outcomes
Palbociclib PALOMA-1 Randomized
phase 2
Aromatase inhibitor
letrozole alone
(standard of care)
versus letrozole
plus palbociclib
Postmenopausal women
with advanced ER+/HER2
breast cancer who had
not received any systemic
treatment for their
advanced disease
Addition of palbociclib markedly
increased median PFS from
10.2 months in the
letrozole group to
20.2 months in the
palbociclib plus
letrozole group
(78) On the basis of this result, palbociclib
received a “Breakthrough Therapy”
designation status from FDA and was
granted accelerated approval, in
combination with letrozole, for the
treatment of ER+/HER2 metastatic
breast cancer
Palbociclib PALOMA-2 Double-blind
phase 3
Palbociclib plus
letrozole as first-
line therapy
Postmenopausal women
with ER+/HER2
breast cancer
Addition of palbociclib strongly
increased median PFS:
14.5 months in the placebo-
letrozole group versus
24.8 months in the
palbociclib-letrozole group
(123) Palbociclib was equally efficacious in
patients with luminal A and B breast
cancers, and there was no single
biomarker associated with the lack of
clinical benefit, except for RB1 loss;
CDK4 amplification was associated
with endocrine resistance, but this
was mitigated by addition of
palbociclib; tumors with high levels
of FGFR2 and ERBB3 mRNA
displayed greater PFS gain
after addition of palbociclib (79)
Palbociclib PALOMA-3 Randomized
phase 3
Estrogen receptor
fulvestrant plus
placebo versus
fulvestrant plus
Women with HR+/HER2
metastatic breast cancer
that had progressed on
previous endocrine therapy
The study demonstrated a
substantial prolongation
of median PFS in the palbociclib-
treated group: 4.6 months in the
placebo plus fulvestrant group
versus 9.5 months in the
palbociclib plus fulvestrant
group; addition of palbociclib
also extended median overall
survival from 28.0 months
(placebo-fulvestrant) to
34.9 months (palbociclib-
fulvestrant); estimated rate
of survival at 3 years was
41% versus 50%, respectively
Palbociclib NeoPalAna Palbociclib
in an
setting (i.e.,
prior to
Compared the effects
of an aromatase
inhibitor anastrozole
versus palbociclib
plus anastrozole
on tumor cell
Women with newly
diagnosed clinical
stage II/III ER+/HER2
breast cancer
Addition of palbociclib enhanced
the antiproliferative effect
of anastrozole
Palbociclib PALLAS Randomized
phase 3
Palbociclib plus
standard endocrine
therapy versus
endocrine therapy
Patients with early
(stage 2 or 3),
breast cancer
Preliminary results indicate that
the trial is unlikely to show
a statistically significant
improvement of invasive
disease-free survival
Palbociclib PENELOPE-B Palbociclib in
patients with
early breast
cancer at high
risk of recurrence
Ribociclib MONA
phase 3
Ribociclib plus
letrozole versus
placebo plus
First-line treatment for
postmenopausal women
with HR+/HER2 recurrent
or metastatic breast
cancer who had not
received previous
systemic therapy for
advanced disease
At 18 months, PFS
was 42.2% in the
group and 63.0%
in the ribociclib-
letrozole group
Ribociclib MONA
Phase 3 Ribociclib plus
Patients with advanced
(metastatic or recurrent)
HR+/HER2 breast cancer
who have either received no
treatment for the advanced
disease or previously
received a single line of
endocrine therapy for the
advanced disease
Addition of ribociclib significantly
extended median PFS, from
12.8 months (placebo-fulvestrant)
to 20.5 months (ribociclib-
fulvestrant); overall survival at
42 months was also extended
from 45.9% (placebo-fulvestrant)
to 57.8% (ribociclib-fulvestrant)
Ribociclib MONA
Phase 3
Ribociclib versus
placebo together
with an anti-
estrogen tamoxifen
or an aromatase
inhibitor (letrozole
or anastrozole)
Premenopausal and
perimenopausal women
with HR+/HER2 advanced
breast cancer who had not
received previous treatment
with CDK4/6 inhibitors
Ribociclib significantly increased
median PFS from 13.0 months in
the placebo-endocrine therapy
group to 23.8 months in the
ribociclib-endocrine therapy
group; overall survival was also
strongly prolonged in the ribociclib
group (estimated overall survival
at 42 months was 46.0% for the
placebo group and 70.2% in the
ribociclib group)
Ribociclib EarLEE-1 Phase 3 trial Ribociclib in the
treatment of early-
stage, high-risk
breast cancers
Abemaciclib MONARCH 1 Phase 2 trial Abemaciclib as a
single agent
Women with HR+/HER2
metastatic breast cancer
who had progressed on or
after prior endocrine therapy
and had 1 or 2 chemotherapy
regimens in the metastatic
Abemaciclib exhibited promising activity
in these heavily pretreated patients
with poor prognosis; median
PFS was 6.0 months and overall
survival 17.7 months
(136) The most common adverse events
were diarrhea, fatigue, and
nausea (136)
Abemaciclib MONARCH 2 Double-blind
phase 3
Abemaciclib in
with fulvestrant
Women with HR+/HER2 breast
cancer who had progressed
while receiving endocrine
therapy, or while receiving
first-line endocrine therapy for
metastatic disease
Addition of abemaciclib significantly
increased PFS from 9.3 months in
the placebo-fulvestrant to 16.4 in
the abemaciclib-fulvestrant group;
median overall survival was also
extended from 37.3 months
to 46.7 months
Abemaciclib MONARCH 3 Randomized
phase 3
Abemaciclib plus
an aromatase
or letrozole)
Postmenopausal women
with advanced HR+/HER2
breast cancer who had
no prior systemic therapy
in the advanced setting
Addition of abemaciclib prolonged
PFS from 14.8 months (in
the placebo-aromatase
inhibitor group) to 28.2 months
inhibitor group)
Abemaciclib MonarchE Phase 3 study Endocrine with
or without
Patients with HR+/HER2
lymph node–positive,
high-risk early
breast cancer
Preliminary analysis indicates that
addition of abemaciclib resulted
in a significant improvement of
invasive disease-free survival
and of distant relapse-
free survival
Trilaciclib Randomized
phase 2 study
Chemotherapy alone
(gemcitabine and
versus concurrent
administration of
trilaciclib plus
administration of
trilaciclib prior to
(to mitigate the
cytotoxic effect of
chemotherapy on
bone marrow)
Patients with recurrent or
metastatic triple-negative
breast cancer who had no
more than two previous
lines of chemotherapy
Addition of trilaciclib did not offer
detectable myeloprotection, but
resulted in increased overall
survival (from 12.8 months in the
chemotherapy-only group to
20.1 months in the concurrent
trilaciclib and chemotherapy
group and 17.8 months in trilaciclib
before chemotherapy group)
(162) The most common adverse events were
neutropenia, thrombocytopenia,
and anemia (162)

Expand for more

Table 3. Major past clinical trials with CDK4/6 inhibitors.
ER+, estrogen receptor–positive; HER2, human epidermal growth factor receptor 2–negative; HR+, hormone receptor–positive; PFS, progression-free survival. FGFR2, fibroblast growth factor receptor 2; ERBB3, receptor tyrosine-protein kinase erbB-3.


Approved by FDA in 2016, in combination with fulvestrant for the treatment of hormone receptor–positive, HER2-negative (HR+/HER2) advanced or metastatic breast cancer in women with disease progression following endocrine therapy. Approved in 2017 for the treatment of HR+/HER2 advanced or metastatic breast cancer in combination with an aromatase inhibitor as initial endocrine-based therapy in postmenopausal women.
Palbociclib is administered at a dose of 125 mg (given orally) daily for 3 weeks followed by 1 week off, or 200 mg daily for 2 weeks followed by 1 week off. The rate-limiting toxicities are neutropenia, thrombocytopenia, and anemia.


Approved by FDA in 2017, in combination with an aromatase inhibitor as initial endocrine-based therapy for the treatment of postmenopausal women with HR+/HER2 advanced or metastatic breast cancer. In 2018, the FDA expanded the indication for ribociclib in combination with an aromatase inhibitor for pre/perimenopausal women with HR+/HER2 advanced or metastatic breast cancer, as initial endocrine-based therapy. FDA also approved ribociclib in combination with fulvestrant for postmenopausal women with HR+/HER2 advanced or metastatic breast cancer, as initial endocrine-based therapy or following disease progression on endocrine therapy.
Ribociclib is administered at a dose of 600 mg (given orally) daily for 3 weeks followed by 1 week off. The main toxicities are neutropenia and thrombocytopenia.


Approved by FDA in 2017, in combination with fulvestrant for women with HR+/HER2 advanced or metastatic breast cancer with disease progression following endocrine therapy. In addition, abemaciclib was approved as monotherapy for women and men with HR+/HER2 advanced or metastatic breast cancer with disease progression following endocrine therapy and prior chemotherapy in the metastatic setting. Approved by FDA in 2018 in combination with an aromatase inhibitor as initial endocrine-based therapy for postmenopausal women with HR+/HER2 advanced or metastatic breast cancer. Approved by FDA in 2021 for adjuvant treatment of early-stage HR+/HER2 breast cancer in combination with endocrine therapy.
Abemaciclib is administered at a dose of 200 mg (given orally) every 12 hours. The dose-limiting toxicity is fatigue. Neutropenia is also observed but is not rate-limiting. Other severe side effects include diarrhea and nausea.
Currently, palbociclib is being used in 164 active or recruiting clinical trials, ribociclib in 69 trials, and abemaciclib in 98 trials for more than 50 tumor types (139). These trials evaluate combinations of CDK4/6 inhibitors with a wide range of compounds (Table 4). Trials with trilaciclib test the benefit of this compound in preserving bone marrow and the immune system.
Additional target Inhibitor Immune
Trial identifier
Aromatase Letrozole, anastrozole,
HR+ breast cancer, HR+ ovarian
cancer, metastatic breast cancer,
metastatic endometrial cancer
LHRH LHRH agonists: goserelin,
HR+ breast cancer NCT03969121,
ER ER antagonists: fulvestrant,
HR+ breast cancer, metastatic
breast cancer
ER Selective estrogen receptor
degraders (SERDs): G1T48,
ZN-c5, SAR439859,
AZD9833, GDC-9545
HR+ breast cancer NCT03455270,
ER Selective estrogen receptor
modulator (SERM):
HR+ breast cancer NCT03820830,
Aromatase + PD-1 Letrozole, anastrozole Pembrolizumab,
Stage IV ER+
breast cancer
PD-1 Nivolumab,
Liposarcoma NCT04438824
PD-L1 Avelumab AR+ breast cancer, TNBC,
ER+/HER2 metastatic
breast cancer
EGFR + PD-L1 Cetuximab Avelumab Squamous cell carcinoma
of the head and neck
HER2 Tucatinib, trastuzumab,
T-DM1, ZW25
HER2+ breast cancer NCT03530696,
EGFR/HER2 Neratinib Advanced solid tumors with
EGFR mutation/amplification,
HER2 mutation/amplification,
HER3/4 mutation, or
KRAS mutation
EGFR Cetuximab Metastatic colorectal cancer,
squamous cell carcinoma
of the head and neck
FGFR Erdafitinib ER+/HER2/FGFR-amplified
metastatic breast cancer
FGFR1-3 Rogaratinib FGFR1-3+/HR+ breast cancer NCT04483505
IGF-1R Ganitumab Ewing sarcoma NCT04129151
VEGF1-3 receptors
+ PD-L1
Axitinib Avelumab NSCLC NCT03386929
RAF Sorafenib Leukemia NCT03132454
MEK PD-0325901,
KRAS and NRAS mutant
metastatic or unresectable
colorectal cancer
ERK Ulixertinib Advanced pancreatic cancer
and other solid tumors
PI3K Copanlisib HR+ breast cancer NCT03128619
PI3K Taselisib, pictilisib,
PIK3CA mutant advanced solid
tumors, PIK3CA mutant and
HR+ breast cancer
PI3K/mTOR Gedatolisib Metastatic breast cancer,
advanced squamous cell lung,
pancreatic, head and neck
cancer and other solid tumors
mTOR Everolimus, vistusertib HR+ breast cancer NCT02871791
AKT Ipatasertib HR+ breast cancer, metastatic
breast cancer, metastatic
gastrointestinal tumors,
BTK Ibrutinib Mantle cell lymphoma NCT03478514
BCL-2 Venetoclax ER+/BCL-2+ advanced
or metastatic breast
AR AR antagonists: bicalutamide AR+ metastatic breast cancer NCT02605486
Lysosome +
Hydroxychloroquine + letrozole ER+ breast cancer NCT03774472
Proliferating cells Standard chemotherapy Stage IV ER+ breast cancer NCT03355157
Proliferating cells Radiation Stage IV ER+ breast cancer NCT03870919,
BCR-ABL Bosutinib HR+ breast cancer NCT03854903
Aromatase Letrozole, anastrozole,
HR+ breast cancer,
metastatic breast
cancer, ovarian
LHRH LHRH agonists:
goserelin, leuprolide
HR+ breast cancer NCT03944434
ER ER antagonists: fulvestrant HR+ breast cancer,
breast cancer
PD-1 Spartalizumab Breast cancer and ovarian
cancer, recurrent and/or
metastatic head and neck
squamous cell carcinoma,
HER2 Trastuzumab, pertuzumab,
HER2+ breast cancer NCT03913234,
EGFR Nazartinib (EGF816) EGFR mutant NSCLC NCT03333343
RAF Encorafenib, LXH254 NSCLC, BRAF
mutant melanoma
MEK Binimetinib BRAF V600-dependent
advanced solid tumors,
PI3K Alpelisib Breast cancer with
PIK3CA mutation
mTOR Everolimus Advanced dedifferentiated
liposarcoma, leiomyosarcoma,
glioma, astrocytoma,
endometrial carcinoma,
pancreatic cancer,
neuroendocrine tumors
mTOR + inflammation Everolimus + dexamethasone ALL NCT03740334
SHP2 TNO155 Advanced solid tumors NCT04000529
AR AR antagonists:
TNBC, metastatic
prostate carcinoma
HDAC Belinostat TNBC, ovarian cancer NCT04315233
proliferating cells Standard chemotherapy Ovarian cancer, metastatic
solid tumors, soft tissue
sarcoma, hepatocellular
Aromatase Letrozole, anastrozole,
HR+ breast cancer,
metastatic breast
cancer, endometrial
ER ER antagonists: fulvestrant Advanced breast cancer,
low-grade serous
ovarian cancer
PD-1 Nivolumab,
Head and neck cancer, g
cancer, NSCLC,
HR+ breast cancer
ER + PD-L1 ER antagonists: fulvestrant Atezolizumab HR+ breast cancer, metastatic
breast cancer
AKT + ER + PD-L1 Ipatasertib + ER
antagonists: fulvestrant
Atezolizumab HR+ breast cancer NCT03280563
PD-L1 LY3300054 Advanced solid tumors NCT02791334
HER2 Trastuzumab HER2+ metastatic
breast cancer
Receptor tyrosine
Sunitinib Metastatic renal
cell carcinoma
IGF-1/IGF-2 Xentuzumab HR+ breast cancer NCT03099174
VEGF-A Bevacizumab Glioblastoma NCT04074785
PI3K Copanlisib HR+ breast cancer, metastatic
breast cancer
PI3K/mTOR LY3023414 Metastatic cancer NCT01655225
ERK1/2 LY3214996 tumors with ERK1/2
mutations, glioblastoma,
metastatic cancer
Proliferating cells Chemotherapy SCLC: This trial evaluates the
potential clinical benefit of
trilaciclib in preventing
myelosuppression in patients
receiving chemotherapy
Proliferating cells +
Carboplatin + etoposide Atezolizumab SCLC: This trial investigates the
potential clinical benefit of trilaciclib
in preserving the bone marrow and
the immune system, and enhancing
antitumor efficacy when
administered with chemotherapy
Proliferating cells Topotecan SCLC: This trial investigates the
potential clinical benefit of
trilaciclib in preserving the
bone marrow and the immune
system, and enhancing the
antitumor efficacy of chemotherapy
when administered prior
to chemotherapy
Proliferating cells Carboplatin + gemcitabine Metastatic TNBC: This study
investigates the potential
clinical benefit of trilaciclib in
preserving the bone marrow
and the immune system, and
enhancing the antitumor efficacy
of chemotherapy when administered
prior to chemotherapy
ER ER antagonist: fulvestrant HR+/HER2 metastatic
breast cancer
EGFR Osimertinib EGFR mutant NSCLC NCT03455829
ER ER antagonist: fulvestrant HR+/HER2 recurrent/
metastatic breast cancer
Aromatase Letrozole, anastrozole HR+/HER2 recurrent/
metastatic breast cancer
EGFR/HER2 Pyrotinib HER2+ gastric cancer, HER2+
metastatic breast cancer
AR AR antagonists: SHR3680 metastatic TNBC NCT03805399
Endocrine therapy Single agent and then
in combination with
endocrine therapy
HR+/HER2 metastatic breast
cancer, ovarian and fallopian tube
cancer, TNBC and other tumors
Aromatase Letrozole ER+/HER2 advanced
breast cancer

Expand for more

Table 4. Ongoing clinical trials testing new combinations with CDK4/6 inhibitors.
HR+, hormone receptor–positive; LHRH, luteinizing hormone–releasing hormone; ER+, estrogen receptor–positive; PD-1, programmed cell death protein 1; PD-L1, programmed cell death 1 ligand 1; AR+, androgen receptor–positive; TNBC, triple-negative breast cancer; EGFR, epidermal growth factor receptor; HER2+, human epidermal growth factor receptor 2–positive; FGFR, fibroblast growth factor receptor; IGFR, insulin-like growth factor receptor; VEGF, vascular endothelial growth factor receptor; PI3K, phosphoinositide 3-kinase; NSCLC, non–small-cell lung cancer; ALL, acute lymphoblastic leukemia; SCLC, small-cell lung cancer.

Resistance to CDK4/6 inhibitors

Although CDK4/6 inhibitors represent very effective agents in cancer treatment, nearly all patients eventually develop resistance and succumb to the disease. Moreover, a substantial fraction of tumors show intrinsic resistance to treatment with CDK4/6 inhibitors (Fig. 3).
Fig. 3. Mechanisms of cancer cell resistance to CDK4/6 inhibition.
Known mechanisms include loss of RB1, activation of pathways impinging on CycD-CDK4/6, amplification of the CDK4/6 genes and overexpression of CDK6 protein, activation of CycE-CDK2, and lysosomal sequestration of CDK4/6 inhibitors. Blank pieces of the puzzle denote additional mechanisms that remain to be discovered.
The best-documented mechanism of preexisting and acquired resistance is the loss of RB1 (7181140). Acquired RB1 loss has been detected in PDXs (141), in circulating tumor DNA (ctDNA) (142143), and in tumors from patients treated with CDK4/6 inhibitors (144145). However, RB1 mutations are likely subclonal and are seen in only 5 to 10% of patients (143145).
Increased expression of CDK6 was shown to underlie acquired resistance to CDK4/6 inhibitors. Amplification of the CDK6 gene and the resulting overexpression of CDK6 protein were found in abemaciclib-resistant ER+ breast cancer cells (146) and in ctDNA of patients with ER+ breast cancers that progressed during treatment with palbociclib plus endocrine therapy (147). Also, CDK4 gene amplification conferred insensitivity to CDK4/6 inhibition in GBM and sarcomas (148150), whereas overexpression of CDK4 protein was associated with resistance to endocrine therapy in HR+ breast cancers (79).
Resistant breast cancer cells can also up-regulate the expression of CDK6 through suppression of the TGF-β/SMAD4 pathway by the microRNA miR-432-5p. In this mechanism, exosomal expression of miR-432-5p mediates the transfer of the resistance phenotype between neighboring cell populations (151). Another mechanism of CDK6 up-regulation in ER+ breast cancers is the loss of FAT1, which represses CDK6 expression via the Hippo pathway. Loss of FAT1 triggers up-regulation of CDK6 expression by the Hippo pathway effectors TAZ and YAP. Moreover, genomic alterations in other components of the Hippo pathway, although rare, are also associated with reduced sensitivity to CDK4/6 inhibitors (81).
Genetic lesions that activate pathways converging on D-type cyclins can cause resistance to CDK4/6 inhibitors. These include (i) FGFR1/2 gene amplification or mutational activation, detected in ctDNA from patients with ER+ breast cancers that progressed upon treatment with palbociclib plus endocrine therapy (147); (ii) hyperactivation of the MAPK pathway in resistant prostate adenocarcinoma cells, possibly due to increased production of EGF by cancer cells (152); and (iii) increased secretion of FGF in palbociclib-resistant KRAS-mutant NSCLC cells, which stimulates FGFR1 signaling in an autocrine or paracrine fashion, resulting in activation of ERK1/2 and mTOR as well as up-regulation of D-cyclin, CDK6, and cyclin E expression (153). Analyses of longitudinal tumor biopsies from a melanoma patient revealed an activating mutation in the PIK3CA gene that conferred resistance to ribociclib plus MEK inhibitor treatment (154). It is possible that these lesions elevate the cellular levels of active cyclin D–CDK4/6 complexes, thereby increasing the threshold for CDK4/6 inhibition.
Formation of a noncanonical cyclin D1–CDK2 complex was shown to represent another mechanism of acquired CDK4/6 inhibitor resistance. Such a complex was observed in palbociclib-treated ER+ breast cancer cells and was implicated in overcoming palbociclib-induced cell cycle arrest (141). Also, depletion of AMBRA1 promoted the interaction of D-cyclins with CDK2, resulting in resistance to CDK4/6 inhibitors (2022); it remains to be seen whether this represents an intrinsic or acquired resistance mechanism in human tumors.
Genetic analyses revealed that activation of cyclin E can bypass the requirement for cyclin D–CDK4/6 in development and tumorigenesis (155156). Hence, it comes as no surprise that increased activity of cyclin E–CDK2 is responsible for a large proportion of intrinsic and acquired resistance to CDK4/6 inhibitors. Several different mechanisms can activate cyclin E–CDK2 kinase in resistant tumor cells: (i) Down-regulation of KIP/CIP inhibitors results in increased activity of cyclin E–CDK (54157). (ii) Loss of PTEN expression, which activates AKT signaling, leads to nuclear exclusion of p27KIP1. This in turn prevents access of p27KIP1 to CDK2, resulting in increased CDK2 kinase activity (144). (iii) Activation of the PI3K/AKT pathway causes decreased levels of p21CIP1. Co-treatment of melanoma PDXs with MDM2 inhibitors (which up-regulate p21CIP1 via p53) sensitized intrinsically resistant tumor cells to CDK4/6 inhibitors (158). (iv) Up-regulation of cyclin D1 levels triggers sequestration of KIP/CIP inhibitors from cyclin E–CDK2 to cyclin D–CDK4/6, thereby activating the former (158). (v) Amplification of the CCNE1 gene and increased levels of cyclin E1 protein result in elevated activity of E-CDK2 kinase (141). (vi) mTOR signaling has been shown to up-regulate cyclin E1 (and D1) in KRAS-mutated pancreatic cancer cells; CDK2 activity was essential for CDK4/6 inhibitor resistance in this setting (159). (vii) Up-regulation of PDK1 results in activation of the AKT pathway, which increases the expression of cyclins E and A and activates CDK2 (160). (viii) In CDK4/6 inhibitor–resistant melanoma cells, high levels of RNA-binding protein FXR1 increase translation of the amino acid transporter SLC36A1. Up-regulation of SLC36A1 expression activates mTORC1, which in turn increases CDK2 expression (161). All these lesions are expected to allow cell proliferation, despite CDK4/6 inhibition, as a consequence of the activation of the downstream cell cycle kinase CDK2.
The role for cyclin E–CDK2 in CDK4/6 inhibitor resistance has been confirmed in clinical trials. In patients with advanced ER+ breast cancer treated with palbociclib and letrozole or fulvestrant, the presence of proteolytically cleaved cytoplasmic cyclin E in tumor tissue conferred strongly shortened progression-free survival (71). Moreover, analyses of PALOMA-3 trial for patients with ER+ breast cancers revealed lower efficacy of palbociclib plus fulvestrant in patients displaying high cyclin E mRNA levels in metastatic biopsies (80). Amplification of the CCNE1 gene was detected in ctDNA of patients with ER+ breast cancers that progressed on palbociclib plus endocrine therapy (147). Also, amplification of the CCNE2 gene (encoding cyclin E2) was seen in a fraction of CDK4/6 inhibitor–resistant HR+ mammary carcinomas (145162).
Collectively, these analyses indicate that resistant cells may become dependent on CDK2 for cell cycle progression. Indeed, depletion of CDK2 or inhibition of CDK2 kinase activity in combination with CDK4/6 inhibitors blocked proliferation of CDK4/6 inhibitor–resistant cancer cells (111141158161). Recently, two CDK2-specific inhibitors, PF-07104091 (163) and BLU0298 (164), have been reported. PF-07104091 is now being tested in a phase 2 clinical trial in combination with palbociclib plus antiestrogens. Another recent study identified a novel compound, PF-3600, that inhibits CDK4/6 and CDK2 (165). PF3600 had potent antitumor effects against xenograft models of intrinsic and acquired resistance to CDK4/6 inhibition (165). A phase 2 clinical trial is currently evaluating this compound as a single agent and in combination with endocrine therapy in patients with HR+/HER2 breast cancer and other cancer types.
Whole-exome sequencing of 59 HR+/HER2 metastatic breast tumors from patients treated with CDK4/6 inhibitors and anti-estrogens revealed eight alterations that likely conferred resistance: RB1 loss; amplification of CCNE2 or AURKA; activating mutations or amplification of AKT1FGFR2, or ERBB2; activating mutations in RAS genes; and loss of ER expression. The frequent activation of AURKA (in 27% of resistant tumors) raises the possibility of combining CDK4/6 inhibitors with inhibitors of Aurora A kinase to overcome resistance (145).
In contrast to ER+ mammary carcinomas, TNBCs are overall resistant to CDK4/6 inhibition (45). A subset of TNBCs display high numbers of lysosomes, which causes sequestration of CDK4/6 inhibitors into the expanded lysosomal compartment, thereby preventing their action on nuclear CDK4/6. Preclinical studies revealed that lysosomotropic agents that reverse the lysosomal sequestration (such as chloroquine, azithromycin, or siramesine) render TNBC cells fully sensitive to CDK4/6 inhibition (71111). These observations now need to be tested in clinical trials for TNBC patients.


Although D-cyclins and CDK4/6 were discovered 30 years ago, several aspects of cyclin D–CDK4/6 biology, such as their role in antitumor immunity, are only now starting to be appreciated. The full range of cyclin D–CDK4/6 functions in tumor cells remains unknown. It is likely that these kinases play a much broader role in cancer cells than is currently appreciated. Hence, the impact of CDK4/6 inhibition on various aspects of tumorigenesis requires further study. Also, treatment of patients with CDK4/6 inhibitors likely affects several aspects of host physiology, which may be relevant to cancer progression.
In the next years, we will undoubtedly witness the development and testing of new CDK4/6 inhibitors. Because activation of CDK2 represents a frequent CDK4/6 inhibitor resistance mechanism, compounds that inhibit CDK4/6 and CDK2 may prevent or delay the development of resistance. Conversely, selective compounds that inhibit CDK4 but not CDK6 may allow more aggressive dosing, as they are expected not to result in bone marrow toxicity caused by CDK6 inhibition. New, less basic CDK4/6 inhibitor compounds (111) may escape lysosomal sequestration and may be efficacious against resistant cancer types such as TNBC. Degrader compounds, which induce proteolysis of cyclin D rather than inhibit cyclin D–CDK4/6 kinase, may have superior properties, as they would extinguish both CDK4/6-dependent and -independent functions of D-cyclins in tumorigenesis. Moreover, dissolution of cyclin D–CDK4/6 complexes is expected to liberate KIP/CIP inhibitors, which would then inhibit CDK2. D-cyclins likely play CDK-independent functions in tumorigenesis—for example, by regulating gene expression (166). However, their role in tumor biology and the utility of targeting these functions for cancer treatment remain largely unexplored.
An important challenge will be to test and identify combinatorial treatments involving CDK4/6 inhibitors for the treatment of different tumor types. CDK4/6 inhibitors trigger cell cycle arrest of tumor cells and, in some cases, senescence. It will be essential to identify combination treatments that convert CDK4/6 inhibitors from cytostatic compounds to cytotoxic ones, which would unleash the killing of tumor cells. Genome-wide high-throughput screens along with analyses of mouse cancer models and PDXs will help to address this point. Another largely unexplored area of cyclin D–CDK4/6 biology is the possible involvement of these proteins in other pathologies, such as metabolic disorders. Research in this area may extend the use of CDK4/6 inhibitors to treatment of other diseases. All these unresolved questions ensure that CDK4/6 biology will remain an active area of basic, translational, and clinical research for several years to come.

CDK inhibitors and Breast Cancer

The U.S. Food and Drug Administration today granted accelerated approval to Ibrance (palbociclib) to treat advanced (metastatic) breast cancer inr postmenopausal women with estrogen receptor (ER)-positive, human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer who have not yet received an endocrine-based therapy. It is to be used in combination with letrozole, another FDA-approved product used to treat certain kinds of breast cancer in postmenopausal women.

See Dr. Melvin Crasto’s blog posts on the announcement of approval of Ibrance (palbociclib) at


and about the structure and mechanism of action of palbociclib



From the CancerNetwork at http://www.cancernetwork.com/aacr-2014/cdk-inhibitors-show-impressive-activity-advanced-breast-cancer

CDK Inhibitors Show Impressive Activity in Advanced Breast Cancer

News | April 08, 2014 | AACR 2014, Breast Cancer

By Anna Azvolinsky, PhD

Ibrance structure


Chemical structure of palbociclib



Palbociclib and LY2835219 are both cyclin-dependent kinase (CDK) 4/6 inhibitors. CDK4 and CDK6 are kinases that, together with cyclin D1, facilitate the transition of dividing cells from the G1 to the S (synthesis) phase of the cell cycle. Preclinical studies have shown that breast cancer cells rely on CDK4 and CDK6 for division and growth, and that selective CDK4/6 inhibitors can arrest the cells at this G1/S phase checkpoint.

The results of the phase II trial of palbociclib and phase I trial of LY2835219 both indicated that hormone receptor (HR)-positive disease appears to be the best marker to predict patient response.

LY2835219 Phase I Trial Demonstrates Early Activity

The CDK4/6 inhibitor LY2835219 has demonstrated early activity in heavily pretreated women with metastatic breast cancer. Nineteen percent of these women (9 out of 47) had a partial response and 51% (24 out of 47) had stable disease following monotherapy with the oral CDK4/6 inhibitor. Patients had received a median of seven prior therapies, and 75% had metastatic disease in the lung, liver, or brain. The median age of patients was 55 years.

All of the partial responses were in patients with HR-positive disease. The overall response rate for this patient subset was 25% (9 of 36 patients). Twenty of the patients with stable disease had HR-positive disease, with 13 patients having stable disease lasting 24 weeks or more.

Despite treatment, disease progression occurred in 23% of the patients.

These results were presented at a press briefing by Amita Patnaik, MD, associate director of clinical research at South Texas Accelerated Research Therapeutics in San Antonio, Texas, at the 2014 American Association for Cancer Research (AACR) Annual Meeting, held April 5–9, in San Diego.

The phase I trial of LY2835219 enrolled 132 patients with five different tumor types, including metastatic breast cancer. Patients received 150-mg to 200-mg doses of the oral drug every 12 hours.

The overall disease control rate was 70% for all patients and 81% among the 36 HR-positive patients.

The median progression-free survival (PFS) was 5.8 months for all patients and 9.1 months for HR-positive patients. Patnaik noted that the median PFS is still a moving target, as 18 patients, all with HR-positive disease, remain on therapy.

“The data are rather encouraging for a very heavily pretreated patient population,” said Patnaik during the press briefing.

Even though the trial was not designed to compare efficacy based on breast cancer subpopulations, the results in HR-positive tumors are particularly encouraging, according to Patnaik.

Common adverse events thought to be treatment-related were diarrhea, nausea, fatigue, vomiting, and neutropenia. These adverse events occurred in 5% or less of patients at grade 3 or 4 toxicity, except neutropenia, which occurred as a grade 3 or 4 toxicity in 11% of patients. Patnaik noted during the press briefing that the neutropenia was uncomplicated and did not result in discontinuation of therapy by any of the patients.

Palbociclib Phase II Data “Impressive”

The addition of the oral CDK4/6 inhibitor palbociclib resulted in an almost doubling of PFS in first-line treatment of postmenopausal metastatic breast cancer patients with HR-positive disease compared with a control population. The patients in this trial were not previously treated for their metastatic breast cancer, unlike the patient population in the phase I LY2835219 trial.

Patients receiving the combination of palbociclib at 125 mg once daily plus letrozole at 2.5 mg once daily had a median PFS of 20.2 months compared with a median of 10.2 months for patients treated with letrozole alone (hazard ratio = 0.488; P = .0004).

Richard S. Finn, MD, assistant professor of medicine at the University of California, Los Angeles, presented the data from the phase II PALOMA-1 trial at a press briefing at the AACR Annual Meeting.

A total of 165 patients were randomized 1:1 to either the experimental arm or control arm.

Forty-three percent of patients in the combination arm had an objective response compared with 33% of patients in the control arm.

Overall survival (OS), a secondary endpoint in this trial, was encouraging but the results are still preliminary, said Finn during the press briefing. The median OS was 37.5 months in the palbociclib arm compared with 33.3 months in the letrozole alone arm (P = .21). Finn noted that long-term follow-up is necessary to establish the median OS. “This first look of the survival data is encouraging. This is a front-line study, and it is encouraging that there is early [separation] of the curves,” he said.

No new toxicities were reported since the interim trial results. Common adverse events included leukopenia, neutropenia, and fatigue. The neutropenia could be quickly resolved and was uncomplicated and not accompanied by fever, said Finn.

Palbociclib is currently being tested in two phase III clinical trials: The PALOMA-3 trial is testing the combination of palbociclib with letrozole and fulvestrant in late-stage metastatic breast cancer patients who have failed endocrine therapy. The PENELOPE-B trial is testing palbociclib in combination with standard endocrine therapy in HR-positive breast cancer patients with residual disease after neoadjuvant chemotherapy and surgery.


  1. Patnaik A, Rosen LS, Tolaney SM, et al. Clinical activity of LY2835219, a novel cell cycle inhibitor selective for CDK4 and CDK6, in patients with metastatic breast cancer. American Association for Cancer Research Annual Meeting 2014; April 5–9, 2014; San Diego. Abstr CT232.
  2. Finn RS, Crown JP, Lang I, et al. Final results of a randomized phase II study of PD 0332991, a cyclin-dependent kinase (CDK)-4/6 inhibitor, in combination with letrozole vs letrozole alone for first-line treatment of ER+/HER2-advanced breast cancer (PALOMA-1; TRIO-18). American Association for Cancer Research Annual Meeting 2014; April 5–9, 2014; San Diego. Abstr CT101.

– See more at: http://www.cancernetwork.com/aacr-2014/cdk-inhibitors-show-impressive-activity-advanced-breast-cancer#sthash.f29smjxi.dpuf


The Cell Cycle and Anti-Cancer Targets




From Cell Cycle in Cancer: Cyclacel Pharmaceuticals™ (note dotted arrows show inhibition of steps e.g. p21, p53)

For a nice video slideshow explaining a bit more on cyclins and the cell cycle please see video below:


Cell Cycle. 2012 Nov 1; 11(21): 3913.

doi:  10.4161/cc.22390

PMCID: PMC3507481

Cyclin-dependent kinase 4/6 inhibition in cancer therapy

Neil Johnson and Geoffrey I. Shapiro*

See the article “Therapeutic response to CDK4/6 inhibition in breast cancer defined by ex vivo analyses of human tumors” in volume 11 on page 2756.

See the article “CDK4/6 inhibition antagonizes the cytotoxic response to anthracycline therapy” in volume 11 on page 2747.

This article has been cited by other articles in PMC.

Cyclin-dependent kinases (CDKs) drive cell cycle progression and control transcriptional processes. The dysregulation of multiple CDK family members occurs commonly in human cancer; in particular, the cyclin D-CDK4/6-retinoblastoma protein (RB)-INK4 axis is universally disrupted, facilitating cancer cell proliferation and prompting long-standing interest in targeting CDK4/6 as an anticancer strategy. Most agents that have been tested inhibit multiple cell cycle and transcriptional CDKs and have carried toxicity. However, several selective and potent inhibitors of CDK4/6 have recently entered clinical trial. PD0332991, the first to be developed, resulted from the introduction of a 2-aminopyridyl substituent at the C2-position of a pyrido(2,3-d)pyrimidin-7-one backbone, affording exquisite selectivity toward CDK4/6.1 PD0332991 arrests cells in G1 phase by blocking RB phosphorylation at CDK4/6-specfic sites and does not inhibit the growth of RB-deficient cells.2 Phase I studies conducted in patients with advanced RB-expressing cancers demonstrated mild side effects and dose-limiting toxicities of neutropenia and thrombocytopenia, with prolonged stable disease in 25% of patients.3,4 In cyclin D1-translocated mantle cell lymphoma, PD0332991 extinguished CDK4/6 activity in patients’ tumors, resulting in markedly reduced proliferation, and translating to more than 1 year of stability or response in 5 of 17 cases.5

Two recent papers from the Knudsen laboratory make several important observations that will help guide the continued clinical development of CDK4/6 inhibitors. In the study by Dean et al., surgically resected patient breast tumors were grown on a tissue culture matrix in the presence or absence of PD0332991. Crucially, these cultures retained associated stromal components known to play important roles in cancer pathogenesis and therapeutic sensitivities, as well as key histological and molecular features of the primary tumor, including expression of ER, HER2 and Ki-67. Similar to results in breast cancer cell lines,6 the authors demonstrate that only RB-positive tumors have growth inhibition in response to PD0332991, irrespective of ER or HER2 status, while tumors lacking RB were completely resistant. This result underscores RB as the predominant target of CDK4/6 in breast cancer cells and the primary marker of drug response in primary patient-derived tumors. As expected, RB-negative tumors routinely demonstrated robust expression of p16INK4A; however, p16INK4A expression was not always a surrogate marker for RB loss, supporting the importance of direct screening of tumors for RB expression to select patients appropriate for CDK4/6 inhibitor clinical trials.

In the second study, McClendon et al. investigated the efficacy of PD0332991 in combination with doxorubicin in triple-negative breast cancer cell lines. Again, RB functionality was paramount in determining response to either PD0332991 monotherapy or combination treatment. In RB-deficient cancer cells, CDK4/6 inhibition had no effect in either instance. However, in RB-expressing cancer cells, CDK4/6 inhibition and doxorubicin provided a cooperative cytostatic effect, although doxorubicin-induced cytotoxicity was substantially reduced, assessed by markers for mitotic catastrophe and apoptosis. Additionally, despite cytostatic cooperativity, CDK4/6 inhibition maintained the viability of RB-proficient cells in the presence of doxorubicin, which repopulated the culture after removal of drug. These results reflect previous data demonstrating that ectopic expression of p16INK4A can protect cells from the lethal effects of DNA damaging and anti-mitotic chemotherapies.7 Similar results have been reported in MMTV-c-neu mice bearing RB-proficient HER2-driven tumors, where PD0332991 compromised carboplatin-induced regressions,8 suggesting that DNA-damaging treatments should not be combined concomitantly with CDK4/6 inhibition in RB-proficient tumors.

To combine CDK4/6 inhibition with cytotoxics, sequential treatment may be considered, in which CDK4/6 inhibition is followed by DNA damaging chemotherapy; cells relieved of G1 arrest may synchronously enter S phase, where they may be most susceptible to agents disrupting DNA synthesis. Release of myeloma cells from a prolonged PD0332991-mediated G1 block leads to S phase synchronization; interestingly, all scheduled gene expression is not completely restored (including factors critical to myeloma survival such as IRF4), further favoring apoptotic responses to cytotoxic agents.9 Furthermore, in RB-deficient tumors, CDK4/6 inhibitors may be used to maximize the therapeutic window between transformed and non-transformed cells treated with chemotherapy. In contrast to RB-deficient cancer cells, RB-proficient non-transformed cells arrested in G1 in response to PD0332991 are afforded protection from DNA damaging agents, thereby reducing associated toxicities, including bone marrow suppression.8

In summary, the current work provides evidence for RB expression as a determinant of response to CDK4/6 inhibition in primary tumors and highlights the complexity of combining agents targeting the cell cycle machinery with DNA damaging treatments.

Go to:


Dean JL, McClendon AK, Hickey TE, Butler LM, Tilley WD, Witkiewicz AK, Knudsen ES. Therapeutic response to CDK4/6 inhibition in breast cancer defined by ex vivo analyses of human tumors Cell Cycle 2012 11 2756 61 doi: 10.4161/cc.21195.

McClendon AK, Dean JL, Rivadeneira DB, Yu JE, Reed CA, Gao E, Farber JL, Force T, Koch WJ, Knudsen ES. CDK4/6 inhibition antagonizes the cytotoxic response to anthracycline therapy Cell Cycle 2012 11 2747 55 doi: 10.4161/cc.21127.

Go to:


Previously published online: www.landesbioscience.com/journals/cc/article/22390

Go to:


  1. Toogood PL, et al. J Med Chem. 2005;48:2388–406. doi: 10.1021/jm049354h. [PubMed] [Cross Ref]
  2. Fry DW, et al. Mol Cancer Ther. 2004;3:1427–38. [PubMed]
  3. Flaherty KT, et al. Clin Cancer Res. 2012;18:568–76. doi: 10.1158/1078-0432.CCR-11-0509. [PubMed] [Cross Ref]
  4. Schwartz GK, et al. Br J Cancer. 2011;104:1862–8. doi: 10.1038/bjc.2011.177. [PMC free article] [PubMed] [Cross Ref]
  5. Leonard JP, et al. Blood. 2012;119:4597–607. doi: 10.1182/blood-2011-10-388298. [PubMed] [Cross Ref]
  6. Dean JL, et al. Oncogene. 2010;29:4018–32. doi: 10.1038/onc.2010.154. [PubMed] [Cross Ref]
  7. Stone S, et al. Cancer Res. 1996;56:3199–202. [PubMed]
  8. Roberts PJ, et al. J Natl Cancer Inst. 2012;104:476–87. doi: 10.1093/jnci/djs002. [PMC free article] [PubMed] [Cross Ref]
  9. Huang X, et al. Blood. 2012;120:1095–106. doi: 10.1182/blood-2012-03-415984. [PMC free article] [PubMed] [Cross Ref]

Cell Cycle. 2012 Jul 15; 11(14): 2756–2761.

doi:  10.4161/cc.21195

PMCID: PMC3409015

Therapeutic response to CDK4/6 inhibition in breast cancer defined by ex vivo analyses of human tumors

Jeffry L. Dean, 1 , 2 A. Kathleen McClendon, 1 , 2 Theresa E. Hickey, 3 Lisa M. Butler, 3 Wayne D. Tilley, 3 Agnieszka K. Witkiewicz, 4 , 2 ,* and Erik S. Knudsen 1 , 2 ,*

Author information ► Copyright and License information ►

See commentary “Cyclin-dependent kinase 4/6 inhibition in cancer therapy” in volume 11 on page 3913.

This article has been cited by other articles in PMC.

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To model the heterogeneity of breast cancer as observed in the clinic, we employed an ex vivo model of breast tumor tissue. This methodology maintained the histological integrity of the tumor tissue in unselected breast cancers, and importantly, the explants retained key molecular markers that are currently used to guide breast cancer treatment (e.g., ER and Her2 status). The primary tumors displayed the expected wide range of positivity for the proliferation marker Ki67, and a strong positive correlation between the Ki67 indices of the primary and corresponding explanted tumor tissues was observed. Collectively, these findings indicate that multiple facets of tumor pathophysiology are recapitulated in this ex vivo model. To interrogate the potential of this preclinical model to inform determinants of therapeutic response, we investigated the cytostatic response to the CDK4/6 inhibitor, PD-0332991. This inhibitor was highly effective at suppressing proliferation in approximately 85% of cases, irrespective of ER or HER2 status. However, 15% of cases were completely resistant to PD-0332991. Marker analyses in both the primary tumor tissue and the corresponding explant revealed that cases resistant to CDK4/6 inhibition lacked the RB-tumor suppressor. These studies provide important insights into the spectrum of breast tumors that could be treated with CDK4/6 inhibitors, and defines functional determinants of response analogous to those identified through neoadjuvant studies.

Keywords: ER, PD0332991, breast cancer, cell cycle, ex vivo

Go to:


Breast cancer is a highly heterogeneous disease.14 Such heterogeneity is known to influence patient response to both standard of care and experimental therapeutics. In regards to biomarker-driven treatment of breast cancers, it was initially recognized that the presence of the estrogen receptor α (ER) in a fraction of breast cancer cells was associated with the response to tamoxifen and similar anti-estrogenic therapies.5,6 Since this discovery, subsequent marker analyses and gene expression profiling studies have further divided breast cancer into a series of distinct subtypes that harbor differing and often divergent therapeutic sensitivities.13 While clearly important in considering the use of several current standard of care therapies, these markers, or molecular sub-types, do not necessarily predict the response to new therapeutic approaches that are currently undergoing clinical development. Thus, there is the continued need for functional analyses of drug response and the definition of new markers that can be used to direct treatment strategies.

Currently, all preclinical cancer models are associated with specific limitations. It is well known that cell culture models lack the tumor microenvironment known to have a significant impact on tumor biology and therapeutic response.79 Xenograft models are dependent on the host response for the engraftment of tumor cells in non-native tissues, which do not necessarily recapitulate the nuances of complex tumor milieu.10 In addition, genetically engineered mouse models, while enabling the tumor to develop in the context of the host, can develop tumors that do not mirror aspects of human disease.10 Furthermore, it remains unclear whether any preclinical model truly represents the panoply of breast cancer subtypes that are observed in the clinic. Herein, we utilized a primary human tumor explant culture approach to interrogate drug response, as well as specific determinants of therapeutic response, in an unselected series of breast cancer cases.

Cell Cycle. 2012 Jul 15; 11(14): 2747–2755.

doi:  10.4161/cc.21127

PMCID: PMC3409014

CDK4/6 inhibition antagonizes the cytotoxic response to anthracycline therapy

  1. Kathleen McClendon, 1 , † Jeffry L. Dean, 1 , † Dayana B. Rivadeneira, 1 Justine E. Yu, 1 Christopher A. Reed, 1 Erhe Gao, 2 John L. Farber, 3 Thomas Force, 2 Walter J. Koch, 2 and Erik S. Knudsen 1 ,*

Author information ► Copyright and License information ►

See commentary “Cyclin-dependent kinase 4/6 inhibition in cancer therapy” in volume 11 on page 3913.

This article has been cited by other articles in PMC.

Go to:


Triple-negative breast cancer (TNBC) is an aggressive disease that lacks established markers to direct therapeutic intervention. Thus, these tumors are routinely treated with cytotoxic chemotherapies (e.g., anthracyclines), which can cause severe side effects that impact quality of life. Recent studies indicate that the retinoblastoma tumor suppressor (RB) pathway is an important determinant in TNBC disease progression and therapeutic outcome. Furthermore, new therapeutic agents have been developed that specifically target the RB pathway, potentially positioning RB as a novel molecular marker for directing treatment. The current study evaluates the efficacy of pharmacological CDK4/6 inhibition in combination with the widely used genotoxic agent doxorubicin in the treatment of TNBC. Results demonstrate that in RB-proficient TNBC models, pharmacological CDK4/6 inhibition yields a cooperative cytostatic effect with doxorubicin but ultimately protects RB-proficient cells from doxorubicin-mediated cytotoxicity. In contrast, CDK4/6 inhibition does not alter the therapeutic response of RB-deficient TNBC cells to doxorubicin-mediated cytotoxicity, indicating that the effects of doxorubicin are indeed dependent on RB-mediated cell cycle control. Finally, the ability of CDK4/6 inhibition to protect TNBC cells from doxorubicin-mediated cytotoxicity resulted in recurrent populations of cells specifically in RB-proficient cell models, indicating that CDK4/6 inhibition can preserve cell viability in the presence of genotoxic agents. Combined, these studies suggest that while targeting the RB pathway represents a novel means of treatment in aggressive diseases such as TNBC, there should be a certain degree of caution when considering combination regimens of CDK4/6 inhibitors with genotoxic compounds that rely heavily on cell proliferation for their cytotoxic effects.



Click on Video Link for Dr. Tolaney slidepresentation of recent data with CDK4/6 inhibitor trial results https://youtu.be/NzJ_fvSxwGk

Audio and slides for this presentation are available on YouTube: http://youtu.be/NzJ_fvSxwGk

Sara Tolaney, MD, MPH, a breast oncologist with the Susan F. Smith Center for Women’s Cancers at Dana-Farber Cancer Institute, gives an overview of phase I clinical trials and some of the new drugs being tested to treat breast cancer. This talk was originally given at the Metastatic Breast Cancer Forum at Dana-Farber on Oct. 5, 2013.

A great article on current clinical trials and explanation of cdk inhibitors by Sneha Phadke, DO; Alexandra Thomas, MD at the site OncoLive




cdk4/6 inhibitor Ibrance Has Favorable Toxicity and Adverse Event Profile


As discussed in earlier posts and the Introduction to this chapter on Cytotoxic Chemotherapeutics, most anti-cancer drugs developed either to target DNA, DNA replication, or the cell cycle usually have similar toxicity profile which can limit their therapeutic use. These toxicities and adverse events usually involve cell types which normally exhibit turnover in the body, such as myeloid and lymphoid and granulocytic series of blood cells, epithelial cells lining the mucosa of the GI tract, as well as follicular cells found at hair follicles. This understandably manifests itself as common toxicities seen with these types of agents such as the various cytopenias in the blood, nausea vomiting diarrhea (although there are effects on the chemoreceptor trigger zone), and alopecia.

It was felt that the cdk4/6 inhibitors would show serious side effects similar to other cytotoxic agents and this definitely may be the case as outlined below:

(Side effects of palbociclib) From navigatingcancer.com

Palbociclib may cause side effects. Tell your doctor if any of these symptoms are severe or do not go away:

  • nausea
  • diarrhea
  • vomiting
  • decreased appetite
  • tiredness
  • numbness or tingling in your arms, hands, legs, and feet
  • sore mouth or throat
  • unusual hair thinning or hair loss

Some side effects can be serious. If you experience any of these symptoms, call your doctor immediately or get emergency medical treatment:

  • fever, chills, or signs of infection
  • shortness of breath
  • sudden, sharp chest pain that may become worse with deep breathing
  • fast, irregular, or pounding heartbeat
  • rapid breathing
  • weakness
  • unusual bleeding or bruising
  • nosebleeds

The following is from FDA Drug Trials Snapshot of Ibrance™:


See PDF on original submission and CDER review

original FDA Ibrance submission

original FDA Ibrance submission

CDER Review Ibrance

CDER Review Ibrance


4.3 Preclinical Pharmacology/Toxicology


For full details, please see Pharmacology/Toxicology review by Dr. Wei Chen The nonclinical studies adequately support the safety of oral administration of palbociclib for the proposed indication and the recommendation from the team is for approval. Non-clinical studies of palbociclib included safety pharmacology studies, genotoxicity

studies, reproductive toxicity studies, pharmacokinetic studies, toxicokinetic studies and repeat-dose general toxicity studies which were conducted in rats and dogs. The pivotal toxicology studies were conducted in compliance with Good Laboratory Practice regulation.



As described above, palbociclib is an inhibitor of CDK4 and CDK6. Palbociclib modulates downstream targets of CDK4 and CDK6 in vitro and induces G1 phase cell cycle arrest and therefore acts to inhibit DNA synthesis and cell proliferation. Combination of palbociclib with anti-estrogen agents demonstrated synergistic inhibition

of cell proliferation in ER+ breast cancer cells. Palbociclib showed anti-tumor efficacy in animal tumor model studies. Safety pharmacology studies with palbociclib demonstrated adverse effects on both the respiratory and cardiovascular function of dogs at a dose of 125mg/day (four times and 50-times the human clinical exposure

respectively) based on mean unbound Cmax.


General toxicology:

Palbociclib was studied in single dose toxicity studies and repeated dose studies in rats and dogs. Adverse effects in the bone marrow, lymphoid tissues, and male reproductive organs were observed at clinically relevant exposures. Partial to complete reversibility of toxicities to the hematolymphopoietic and male reproductive systems was demonstrated following a recovery period (4-12 weeks), with the exception of the male reproductive organ findings in dogs. Gastrointestinal, liver, kidney, endocrine/metabolic (altered glucose metabolism), respiratory, ocular, and adrenal effects were also seen.


Genetic toxicology:

Palbociclib was evaluated for potential genetic toxicity in in vitro and in vivo studies. The Ames bacterial mutagenicity assay in the presence or absence of metabolic activation demonstrated non-mutagenicity. In addition, palbociclib did not induce chromosomal aberrations in cultured human peripheral blood lymphocytes in the presence or absence of metabolic activation. Palbociclib was identified as aneugenic based on kinetochore analysis of micronuclei formation in an In vitro assay in CHO-WBL cells. In addition, palbociclib was shown to induce micronucleus formation in male rats at doses 100

mg/kg/day (10x human exposure at the therapeutic dose) in an in vivo rat micronucleus assay.


Reproductive toxicology: No effects on estrous cycle and no reproductive toxicities were noticed in standard assays.


Pharmacovigilance (note please see PDF for more information)

Deaths Associated With Trials: Although a few deaths occurred during some trials no deaths were attributed to the drug.

Non-Serious Adverse Events:

(note a reviewers comment below concerning incidence of pulmonary embolism is a combination trial with letrazole)


fda ibrance reviewers SAE comment


Other article in this Open Access Journal on Cell Cycle and Cancer Include:


Tumor Suppressor Pathway, Hippo pathway, is responsible for Sensing Abnormal Chromosome Numbers in Cells and Triggering Cell Cycle Arrest, thus preventing Progression into Cancer

Nonhematologic Cancer Stem Cells [11.2.3]

New methods for Study of Cellular Replication, Growth, and Regulation

Multiple Lung Cancer Genomic Projects Suggest New Targets, Research Directions for Non-Small Cell Lung Cancer

Proteomics, Metabolomics, Signaling Pathways, and Cell Regulation: a Compilation of Articles in the Journal http://pharmaceuticalintelligence.com

In Focus: Targeting of Cancer Stem Cells









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Curation of Recently Halted Oncology Trials Due to Serious Adverse Events – 2015

Curator: Stephen J. Williams, Ph.D.

The following is reports of oncology clinical trials in 2015 which have been halted for Serious Adverse Events (SAE), in most instances of an idiopathic nature. For comparison I have listed (as of this writing) the oncology drug approvals (8) for 2015. (from CenterWatch.com)

Oncology Drugs Approved in 2015

Farydak (panobinostat); Novartis; For the treatment of multiple myeloma, Approved February 2015

Ibrance (palbociclib); Pfizer; For the treatment of ER-positive, HER2-negative breast cancer, Approved February 2015

Lenvima (lenvatinib); Eisai; For the treatment of thyroid cancer, Approved February 2015

Lonsurf (trifluridine and tipiracil); Taiho Oncology; For the treatment of metastatic colorectal cancer , Approved September 2015

Odomzo (sonidegib); Novartis; For the treatment of locally advanced basal cell carcinoma, July 2015

Opdivo (nivolumab); Bristol-Myers Squibb; For the treatment of metastatic squamous non-small cell lung cancer, Approved March 2015

Unituxin (dinutuximab); United Therapeutics; For the treatment of pediatrics with high-risk neuroblastoma, Approved March 2015

Varubi (rolapitant); Tesaro; For the prevention of delayed nausea and vomiting associated with chemotherapy, Approved September 2015

Death Forces FDA to Place Clinical Hold on Advaxis (ADXS) Cancer Drug

from Biospace News

October 7, 2015
By Alex Keown, BioSpace.com Breaking News Staff

PRINCETON, N.J. – Following the death of a patient, the U.S. Food and Drug Administration (FDA) placed a hold on Advaxis (ADXS)’s experimental cancer treatment axalimogene filolisbac, which is currently in mid-stage trials.

In a statement issued this morning, Advaxis maintains the patient’s death was a result of the severity of her cancer and not due to the company’s experimental cancer treatment. It is seeking proof from the FDA that the drug was not a factor in the death. Still, the hold on the experimental cancer drug will cause the company to halt four clinical trials, Advaxis said. Other clinical trials, including those with the experimental ADXS-PSA and ADXS-HER2, are not affected by this hold. The company said it will continue to actively enroll and dose patients.

The FDA placed a hold on the drug on Oct. 2 after the company submitted a safety report to the regulatory agency that week. The drug is being developed to treat patients with persistent or recurrent metastatic (squamous or non-squamous cell) carcinoma of the cervix (PRmCC) who have progressed on at least one prior line of systemic therapy. Phase I trials released at the end of September showed treatment with axalimogene filolisbac resulted in a 38.5 percent 12-month overall survival rate in 26 patients. Patients typically fighting PRmCC who have failed at least one line of therapy have a typical survival rate of four to seven months.

Read full story here

FDA Halts Trial of Halozyme’s PEGPH20 for Pancreatic Cancer

Apr 9, 2014 Alex Philippidis

Halozyme Therapeutics acknowledged today that the FDA placed a formal clinical hold on its troubled Study 202 assessing its experimental drug PEGPH20 in patients with pancreatic cancer—less than a week after the company temporarily halted enrolling and dosing patients in the ongoing Phase II trial.

The agency told Halozyme it placed the clinical hold following the company’s pause in study activity. The trial’s independent data monitoring committee is evaluating data from the trial to learn why patients treated with PEGPH20 as well as nab-paclitaxel and gemcitabine saw a higher rate of blood clots and other thromboembolic events compared with patients treated with nab-paclitaxel and gemcitabine alone.

“We will be providing this information to the data monitoring committee and the FDA in parallel so they can complete their respective assessments,” Helen Torley, M.B. Ch.B., M.R.C.P., Halozyme’s president and CEO, said in a statement.

“Pancreatic cancer has one of the lowest survival rates of any cancer. We remain committed to evaluating PEGPH20 as a possible therapy to address this devastating disease,” Dr. Torley added.

As with Halozyme’s statement last week, the company’s latest remarks did not indicate when Halozyme expects to resume enrolling and dosing patients in Study 202, or how many patients had been enrolled and dosed when the temporary halt occurred.

The trial was envisioned as having 124 subjects, divided evenly between a treatment arm of PEGPH20 and nab-paclitaxel, and a gemcitabine arm, preceded by eight subject “run-in” phase assessing safety and tolerability, according to Study 202’s page on ClinicalTrials.gov (NCT01839487), last updated on January 27.

The study is one of two Phase II trials for PEGPH20; the other, SWOG, also aims to assess the drug for pancreatic cancer.

PEGPH20 is an investigational PEGylated form of Halozyme’s FDA-approved recombinant human hyaluronidase rHuPH20 (marketed as Hylenex®), designed to dramatically increases the half-life of the compound in the blood and allow for intravenous administration.

The temporary halt for Study 202 came two months after Halozyme publicly cited “potential acceleration of the PEGPH20 program” among several R&D programs for which it raised funds through a public offering of common stock that closed in February and generated approximately $107.8 million in net proceeds.

Read more at GenNEWS

FDA orders CytRx to halt patient enrollment after death of a cancer patient

CytRx ($CYTR) has run into an unexpected roadblock with its cancer drug conjugate aldoxorubicin, slamming the brakes on new patient recruitment in all their clinical trials after the FDA dropped a partial clinical hold on the program. According to the biotech the hold was forced by the death of a patient who was given the drug through a compassionate use program.

LA-based CytRx execs say that patients already enrolled in the studies will continue to receive the therapy as investigators added new safety measures, retooling trial protocols to include an “appropriate inclusion/exclusion criteria, an additional patient screening assessment and an evaluation of serum electrolytes prior to aldoxorubicin administration.” The patient who died, they added, had not qualified for any of its studies.

As it stands now, the biotech doesn’t know exactly how long the partial hold will last, but their announcement sought to calm jumpy investors, saying they expected to resolve the FDA’s demands “expeditiously” and can stick to their current timelines. CytRx says it expects to report preliminary results from their mid-stage study of Kaposi’s sarcoma in the second quarter of 2015 and preliminary results from the ongoing Phase II clinical trial of aldoxorubicin in glioblastoma multiforme in the first half of 2015. The company added that it is committed to completing enrollment in their Phase III trial by the end of next year.

hat reassurance appears to have helped with investors, who seemed to count this as more of a temporary setback than a catastrophe. Shares for CytRx were down about 9% in mid-morning trading.

Aldoxorubicin uses a linker molecule to attach to albumin in the blood and concentrate in tumors, where the acidic environment releases the chemotherapy doxorubicin in doses up to four times higher than what’s used now. Late last year their stock soared after their drug scored promising results for progression-free survival in a Phase IIb trial.

This case illustrates one reason why biotechs often quietly squirm under the pressure of compassionate use programs. They can be expensive to operate, time-consuming and raise fresh concerns when a patient dies or experiences a setback. On the other hand, if regulators take action like this following the death of an advanced stage cancer patient, there may have been something about the case that triggered broader concerns for the entire patient population

Clot risk in Lilly lung-cancer drug raises FDA concerns

July 7, 2015

Eli Lilly and Co.’s experimental lung cancer drug has raised concerns with U.S. regulators that it may increase patients’ risk of suffering potentially deadly blood clots.

The drug, known as necitumumab, improved patients’ overall chances of survival, yet people taking the medicine also experienced more risk, Food and Drug Administration staff said in a report Tuesday. Indianapolis-based Lilly is seeking to sell the medicine to treat a subset of the most common type of lung cancer.

FDA advisers will meet Thursday to discuss the risks and benefits of necitumumab for patients with advanced squamous non-small cell lung cancer, in combination with chemotherapy. The FDA is expected to decide if Lilly can sell the drug by the end of the year.

While the safety of necitumumab reflects that of similar drugs, the increased danger of clotting “in this already high risk population is of concern,” FDA staff wrote.

One study showed that out of 538 patients taking necitumumab and chemotherapy, 9 percent experienced a serious clot, compared with 5 percent of 541 patients given only chemotherapy, according to the staff report.

Squamous lung cancer accounts for 25 percent to 30 percent of all lung cancer, according to the American Cancer Society.

Patients in a clinical trial who took necitumumab lived a median of 11.5 months, 1.6 months longer than those who got only chemotherapy, the FDA staff report said.

Opdivo Side Effects Center (as seen on Rxlist.com) (NOTE:TRIAL NOT HALTED)

Last reviewed on RxList 10/05/2015

Opdivo (nivolumab) is a human monoclonal antibody used to treat patients with unresectable or metastatic melanoma and disease progression following ipilimumab and, if BRAF V600 mutation positive, a BRAF inhibitor; and to treat metastatic squamous non-small cell lung cancer with progression on or after platinum-based chemotherapy. Common side effects of Opdivo include fatigue, rash, itching, cough, upper respiratory tract infection, swelling of the extremities, shortness of breath, muscle pain, decreased appetite, nausea, vomiting, constipation, diarrhea, weakness, swelling, fever, abdominal pain, chest pain, joint pain, and weight loss.

Opdivo FDA Prescribing Information: Side Effects
(Adverse Reactions)

Clinical Trials Experience

Because clinical trials are conducted under widely varying conditions, adverse reaction rates observed in the clinical trials of a drug cannot be directly compared to rates in the clinical trials of another drug and may not reflect the rates observed in clinical practice.

The data described in the WARNINGS AND PRECAUTIONS section and below reflect exposure to OPDIVO in Trial 1, a randomized trial in patients with unresectable or metastatic melanoma and in Trial 3, a single-arm trial in patients with metastatic squamous non-small cell lung cancer (NSCLC).

Clinically significant adverse reactions were evaluated in a total of 691 patients enrolled in Trials 1, 3, or an additional dose finding study (n=306) administering OPDIVO at doses of 0.1 to 10 mg/kg every 2 weeks [see WARNINGS AND PRECAUTIONS].

Unresectable or Metastatic Melanoma

The safety of OPDIVO was evaluated in Trial 1, a randomized, open-label trial in which 370 patients with unresectable or metastatic melanoma received OPDIVO 3 mg/kg every 2 weeks (n=268) or investigator’s choice of chemotherapy (n=102), either dacarbazine 1000 mg/m² every 3 weeks or the combination of carboplatin AUC 6 every 3 weeks plus paclitaxel 175 mg/m² every 3 weeks [see Clinical Studies]. The median duration of exposure was 5.3 months (range: 1 day to 13.8+ months) with a median of eight doses (range: 1 to 31) in OPDIVO-treated patients and was 2 months (range: 1 day to 9.6+ months) in chemotherapy treated patients. In this ongoing trial, 24% of patients received OPDIVO for greater than 6 months and 3% of patients received OPDIVO for greater than 1 year.

In Trial 1, patients had documented disease progression following treatment with ipilimumab and, if BRAF V600 mutation positive, a BRAF inhibitor. The trial excluded patients with autoimmune disease, prior ipilimumab-related Grade 4 adverse reactions (except for endocrinopathies) or Grade 3 ipilimumab-related adverse reactions that had not resolved or were inadequately controlled within 12 weeks of the initiating event, patients with a condition requiring chronic systemic treatment with corticosteroids ( > 10 mg daily prednisone equivalent) or other immunosuppressive medications, a positive test for hepatitis B or C, and a history of HIV.

The study population characteristics in the OPDIVO group and the chemotherapy group were similar: 66% male, median age 59.5 years, 98% white, baseline ECOG performance status 0 (59%) or 1 (41%), 74% with M1c stage disease, 73% with cutaneous melanoma, 11% with mucosal melanoma, 73% received two or more prior therapies for advanced or metastatic disease, and 18% had brain metastasis. There were more patients in the OPDIVO group with elevated LDH at baseline (51% vs. 38%).

OPDIVO was discontinued for adverse reactions in 9% of patients. Twenty-six percent of patients receiving OPDIVO had a drug delay for an adverse reaction. Serious adverse reactions occurred in 41% of patients receiving OPDIVO. Grade 3 and 4 adverse reactions occurred in 42% of patients receiving OPDIVO. The most frequent Grade 3 and 4 adverse reactions reported in 2% to less than 5% of patients receiving OPDIVO were abdominal pain, hyponatremia, increased aspartate aminotransferase, and increased lipase.

FDA Approves Eisai’s LENVIMA™ (lenvatinib) for the Treatment of Patients with Locally Recurrent or Metastatic, Progressive, Radioactive Iodine-Refractory Differentiated Thyroid Cancer

– Press release from Eisai (NOTE: TRIAL NOT HALTED)

Feb 13, 2015

WOODCLIFF LAKE, N.J., Feb. 13, 2015 /PRNewswire/ — Eisai Inc. announced today that the U.S. Food and Drug Administration (FDA) approved the company’s receptor tyrosine kinase inhibitor LENVIMA™ (lenvatinib) for the treatment of locally recurrent or metastatic, progressive, radioactive iodine-refractory differentiated thyroid cancer (RAI-R DTC). LENVIMA was approved following a priority review by the FDA, which is designated for drugs the FDA believes have the potential to provide a significant improvement in the treatment of a serious condition. LENVIMA demonstrated a statistically significant progression-free survival (PFS) prolongation and response rate in patients with progressive, differentiated thyroid cancer who had become refractory to radioactive iodine (RAI) therapy.

In the clinical trial, adverse events led to dose reductions in 68% of patients who received LENVIMA and 5% of patients who received placebo. Some patients will need to discontinue treatment for serious adverse reactions. In the trial, 18% of patients treated with LENVIMA and 5% who received placebo discontinued treatment. The most common adverse reactions (at least 10%) that resulted in dose reductions of LENVIMA were hypertension (13%), proteinuria (11%), decreased appetite (10%), and diarrhea (10%).

AstraZeneca halts a pair of lung cancer trials over a safety scare

From October 9, 2015 | By of FierceBiotech

“AstraZeneca ($AZN) is pressing pause on trials combining two of its most important pipeline cancer treatments after tracking reports of lung disease, halting enrollment as it gathers more information.

The company is testing a combination of AZD9291 and durvalumab, formerly MEDI4736, in two studies involving patients with non-small cell lung cancer. Late last month, AstraZeneca hit the brakes on enrollment in both trials due to an increase in reports of interstitial lung disease, which can lead to dangerous scarring and impaired pulmonary function. The pauses are temporary, the company stressed in an emailed statement, and patients already enrolled in the study will be given new consent forms to ensure they understand the risks before choosing whether keep getting treatment.”

Other posts on this site on Cytotoxicity and Cancer include

Novel Approaches to Cancer Therapy [11.1]

Misfolded Proteins – from Little Villains to Little Helpers… Against Cancer

Multiple Lung Cancer Genomic Projects Suggest New Targets, Research Directions for Non-Small Cell Lung Cancer

A Synthesis of the Beauty and Complexity of How We View Cancer

Good and Bad News Reported for Ovarian Cancer Therapy

Read Full Post »

Steroids, Inflammation, and CAR-T Therapy [6.3.8] (UPDATED)

Reporter: Stephen J. Williams, Ph.D.

UPDATED: 08/31/2020 (CRISPR edited CAR-T clinical trials)

Word Cloud by Daniel Menzin

Corticosteroids have been used as anticancer agents since the 1940s, with activity reported in a wide variety of solid tumors, including breast and prostate cancer, and the lymphoid hematologic malignancies. They are commonly found in regimens for acute lymphocytic leukemia, Hodgkin’s and non-Hodgkin’s lymphoma, myeloma, and chronic lymphocytic leukemia.

A great review on the mechanism of action of prednisone’s antitumoral activity is seen in

Corticosteroids in the Treatment of Neoplasms Lorraine I. McKay, PhD and John A. Cidlowski, PhD. in Holland-Frei Cancer Medicine. 6th edition.

It was first discovered that cortisone caused tumor regression in a transplantable mouse lymphosarcoma,81 a finding soon extended to a wide variety of murine lymphatic tumors. The effects of corticosteroids were also evaluated on many nonendocrine and nonlymphoid transplantable rodent tumors. Pharmacologic doses of steroid inhibited growth of various tumor systems.82 Tissue culture studies confirmed that lymphoid cells were the most sensitive to glucocorticoids, and responded to treatment with decreases in DNA, ribonucleic acid (RNA), and protein synthesis.83 Studies of proliferating human leukemic lymphoblasts supported the hypothesis that glucocorticoids have preferential lymphocytolytic effects. The mechanism of action was initially thought to be caused by impaired energy use via decreased glucose transport and/or phosphorylation; it was later discovered that glucocorticoids induce apoptosis, or programmed cell death, in certain lymphoid cell populations.84,85

–For review on corticosteroids in cancer therapy see more at: http://www.cancernetwork.com/review-article/corticosteroids-advanced-cancer#sthash.IwHbekuI.dpuf

However, as Dana Farber’s Dr. George Canellos, M.D. ponders in Can MOPP be replaced in the treatment of advanced Hodgkin’s disease? Semin Oncol. Canellos GP1. 1990 Feb;17(1 Suppl 2):2-6., many dose-limiting toxicities occur with MOPP (mechlorethamine, vincristine, procarbazine, prednisone) therapy used in advanced Hodgkin’s disease.  Although, at the time, he generally was looking to establish combination therapies with less side effect, the advent of more personalized therapies as well as immunotherapies may indeed replace the older regimens for B-cell malignancies and Hodgkin’s disease, and their panels of toxicities.

Short-term side effects of prednisone (Cancer.gov prednisone description with side effects) as with all glucocorticoids, include high blood glucose levels (especially in patients with diabetes mellitus or on other medications that increase blood glucose, such as tacrolimus) and mineralocorticoid effects such as fluid retention.[10] The mineralocorticoid effects of prednisone are minor, which is why it is not used in the management of adrenal insufficiency, unless a more potent mineralocorticoid is administered concomitantly.

Long-term side effects include Cushing’s syndrome, steroid dementia syndrome, truncal weight gain, osteoporosis, glaucoma and cataracts, type II diabetes mellitus, and depression upon dose reduction or cessation.

Therefore the oncology world has been moving toward therapies which are more selective with less dose-limiting toxicities (e.g. Rituximab), and are looking to CAR-T therapies as a possible replacement for standard chemotherapeutic regimens. However, as with prednisone, there have been serious adverse events in some CAR-T clinical trials. Luckily clinicians, as discussed below, have found supportive therapies to alleviate the most severe side effects to CAR-T.

This section will be refer to supportive therapies as those adjuvant therapy given to alleviate patient discomfort, reduce toxicities and adverse event, or support patient well-being during their course of chemotherapy, not adjuvant therapy to enhance antitumoral effect.

For more background information of CAR-T therapies and related issues please see my previous post

NIH Considers Guidelines for CAR-T therapy: Report from Recombinant DNA Advisory Committee

The following is a brief re-post of some of the important points for reference to this new posting.

1. Evolution of Chimeric Antigen Receptors

Early evidence had suggested that adoptive transfer of tumor-infiltrating lymphocytes, after depletion of circulating lymphocytes, could result in a clinical response in some tumor patients however developments showed autologous T-cells (obtained from same patient) could be engineered to express tumor-associated antigens (TAA) and replace the TILS in the clinical setting.

A brief history of construction of 2nd and 3rd generation CAR-T cells given by cancer.gov:



Differences between  second- and third-generation chimeric antigen receptor T cells. (Adapted by permission from the American Association for Cancer Research: Lee, DW et al. The Future Is Now: Chimeric Antigen Receptors as New Targeted Therapies for Childhood Cancer. Clin Cancer Res; 2012;18(10); 2780–90. doi:10.1158/1078-0432.CCR-11-1920)

Constructing a CAR T Cell (from cancer.gov)

The first efforts to engineer T cells to be used as a cancer treatment began in the early 1990s. Since then, researchers have learned how to produce T cells that express chimeric antigen receptors (CARs) that recognize specific targets on cancer cells.

The T cells are genetically modified to produce these receptors. To do this, researchers use viral vectors that are stripped of their ability to cause illness but that retain the capacity to integrate into cells’ DNA to deliver the genetic material needed to produce the T-cell receptors.

The second- and third-generation CARs typically consist of a piece of monoclonal antibody, called a single-chain variable fragment (scFv), that resides on the outside of the T-cell membrane and is linked to stimulatory molecules (Co-stim 1 and Co-stim 2) inside the T cell. The scFv portion guides the cell to its target antigen. Once the T cell binds to its target antigen, the stimulatory molecules provide the necessary signals for the T cell to become fully active. In this fully active state, the T cells can more effectively proliferate and attack cancer cells.

2. Consideration for Design of Trials and Mitigating Toxicities

  • Early Toxic effectsCytokine Release Syndrome– The effectiveness of CART therapy has been manifested by release of high levels of cytokines resulting in fever and inflammatory sequelae. One such cytokine, interleukin 6, has been attributed to this side effect and investigators have successfully used an IL6 receptor antagonist, tocilizumab (Acterma™), to alleviate symptoms of cytokine release syndrome (see review Adoptive T-cell therapy: adverse events and safety switches by Siok-Keen Tey).
  • Early Toxic effects – Over-activation of CAR T-cells; mitigation by dose escalation strategy (as authors in reference [3] proposed). Most trials give billions of genetically modified cells to a patient.
  • Late Toxic Effectslong-term depletion of B-cells . For example CART directing against CD19 or CD20 on B cells may deplete the normal population of CD19 or CD20 B-cells over time; possibly managed by IgG supplementation


  1. Ertl HC, Zaia J, Rosenberg SA, June CH, Dotti G, Kahn J, Cooper LJ, Corrigan-Curay J, Strome SE: Considerations for the clinical application of chimeric antigen receptor T cells: observations from a recombinant DNA Advisory Committee Symposium held June 15, 2010. Cancer research 2011, 71(9):3175-3181.
  2. Morgan RA, Yang JC, Kitano M, Dudley ME, Laurencot CM, Rosenberg SA: Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Molecular therapy : the journal of the American Society of Gene Therapy 2010, 18(4):843-851.
  3. Kandalaft LE, Powell DJ, Jr., Coukos G: A phase I clinical trial of adoptive transfer of folate receptor-alpha redirected autologous T cells for recurrent ovarian cancer. Journal of translational medicine 2012, 10:157.

3. Case Reports of Adverse Events and Their Amelioration During CAR-T Therapy

CAR-T Therapy have Had reports of Serious Adverse Events

From FierceBiotech UPDATED: Two deaths force MSK to hit the brakes on engineered T cell cancer study

April 6, 2014 | By John Carroll

Safety concerns forced investigators at Memorial Sloan-Kettering Cancer Center to suspend patient recruitment for an early-stage study of a closely watched approach to reengineering the immune system to fight cancer. Several days ago MSK updated a site on clinicaltrials.gov to note that it was halting recruitment for a small study using T cells reengineered with chimeric antigen receptors (CARs) against CD19-positive B cells for aggressive non-Hodgkin lymphoma, triggering concerns about the potential fallout at Juno Therapeutics, the biotech formed to commercialize the effort. And Sunday evening representatives for MSK revealed at the meeting of the American Association for Cancer Research in San Diego that the deaths of two patients spurred investigators to rethink the trial protocol on recruitment, revamping the patient profile to account for the threat of comorbidities while adjusting the dose “based on the extent of disease at the time of treatment.”

For more on this story please see

Source: http://www.fiercebiotech.com/story/memorial-sloan-kettering-hits-brakes-engineered-t-cell-cancer-study/2014-04-06

Keynote presentation by Carl H. June, recipient of The Richard V. Smalley MD 2013 Award

As reported in 2013 in Highlights and summary of the 28th annual meeting of the Society for Immunotherapy of Cancer by Paolo A Ascierto1, David H Munn2, Anna K Palucka34 and Paul M Sondel in Journal of ImmunoTherapy of Cancer

Since 2005, SITC honors a luminary in the field who has significantly contributed to the advancement of cancer immunotherapy research by presenting the annual Richard V. Smalley MD Memorial Award, which is associated with the Smalley keynote lecture at the Annual SITC meeting. The awardee this year Carl H. June of the University of Pennsylvania, has led innovative translational research for over 25 years, with the most recent focus being the development of the Chimeric Antigen Receptor modified T-cell (CART) approach. Carl June summarized how the past 15 years of progress have expanded upon the original concept presented by Zelig Eshhar [4], in which variable regions of tumor-reactive monoclonal antibodies (mAbs) (VH and VL) are linked to transmembrane and signaling domains of T cell activating molecules to create membrane based receptors with specificity for the tumor antigen recognized by the original mAb [4]. These receptors can be transfected into T cells, for example with lentiviruses. Pre-clinical work demonstrated how CD3-ζ and 41BB signaling components enhanced proliferation and survival of T cells in hypoxic conditions. The initial clinical work has been done with CART reactive to CD-19 on malignant B cells, with progress particularly in chronic lymphocytic leukemia (CLL) in adults and acute lymphoblastic leukemia (ALL) in children [5,6]. As of the SITC meeting, CarlJune’s team had treated 35 patients with CLL and 20 with ALL. Of the 20 with ALL, ½ had relapsed after allogeneic BMT. Of these 20 children, 17 were in complete remission, and with persistent B cell aplasia; documenting the persistent effects of the CART cells. Toxicities included the persistent B cell aplasia and profound tumor lysis and cytokine storm, seen 1–2 weeks into the treatment for ALL. This cytokine storm has been ameliorated by using anti-IL6 mAb. The B cell aplasia, while undesired, is acceptable, as patients can receive passive replacement of IgG, thus making their B cells “expendable”. These CART cells can traffic into the CNS. In ALL patients, it appears that each individual CART cell (or its progeny) can destroy 1000 tumor cells. Ongoing efforts in CarlJune’s program, and at other centers, are now moving into analyses of CART reactive with other tumor targets, by using mAbs that recognize antigens expressed on other tumors. Among these are EGFR on glioblastoma, PSMA on prostate cancer, mesothelin on ovarian cancer, HER2 on breast (and other) cancers, and several other targets. Because some of these targets are also expressed on normal tissues that are “not expendable”, novel approaches are being developed to decrease the potency or longevity of the CART effect, to decrease potential toxicity. This includes generating “short lived” CART cells by inducing CAR expression with short-lived RNA, rather than transfecting with a DNA construct that remains permanently.

In T-Cell Immunotherapy: Looking Forward Molecular Therapy (2014); 22 9, 1564–1574. doi:10.1038/mt.2014.148 many of the leading CAR-T clinicians and investigators reported on some of the adverse events related o CAR-T therapy including

  • 40 severe adverse events (SAE) had been reported from 2010 to 2013.
  • B-cell aplasia
  • Systemic inflammatory release syndrome (CRS) {the most sever toxicity seen}
  • Tumor lysis syndrome
  • CNS toxicity
  • Macrophage activation syndrome

According to the investigators the systemic inflammatory release syndrome (CRS) is the most severe toxicity seen

The most commonly reported adverse event is CRS,49 with about three-quarters of the patients with CRS requiring admission to an intensive care unit. In the case of CAR therapy, the onset of CRS is related to the particular signaling domain in the CAR, with early-onset CRS in the first several days after infusion related to CARs that encode a CD28 signaling domain.4,16 By contrast, CARs encoding a 4-1BB signaling domain tend to have delayed-onset CRS (range, 7 to 50 days) after CAR T-cell infusion.6 CRS has also been reported after the infusion of TCR-modified T cells, with onset typically five to seven days after infusion. The development of CRS is often, but not invariably, associated with clinically beneficial tumor regression. Several cytokines have been reported to be elevated in the serum—most commonly, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-6. Management of CRS has included supportive care, corticosteroids, etanercept, tocilizumab, and alemtuzumab. The role of suicide genes in the management of CRS remains unknown.50

This supportive therapy have now been included in all protocols now and sites are engaged in developing pharmacovigilance protocol development for CAR-T therapy.

UPDATED 08/31/2020

The following articles discuss the use of the CRISPR Cas9 system to improve the efficacy and reduce immune tolerance and rejection of engineered CAR-T therapies and the evolution of the CAR-T therapy in clinical trials for lung cancer and leukemias.

CRISPR-engineered immune cells reach the bedside

UPDATED 10/04/2021 (Advances in CAR-T therapy for solid tumors)

The following contains a curation of the latest advances on CAR-T therapy for solid tumors, which has been a challenge for the field.

NK cells expressing a chimeric activating receptor eliminate MDSCs and rescue impaired CAR-T cell activity against solid tumors

From: Parihar, R., Rivas, C., Huynh, M., Omer, B., Lapteva, N., Metelitsa, L. S., Gottschalk, S. M., & Rooney, C. M. (2019). NK Cells Expressing a Chimeric Activating Receptor Eliminate MDSCs and Rescue Impaired CAR-T Cell Activity against Solid Tumors. Cancer immunology research7(3), 363–375. https://doi.org/10.1158/2326-6066.CIR-18-0572


Solid tumors are refractory to cellular immunotherapies in part because they contain suppressive immune effectors such as myeloid-derived suppressor cells (MDSCs) that inhibit cytotoxic lymphocytes. Strategies to reverse the suppressive tumor microenvironment (TME) should also attract and activate immune effectors with antitumor activity. To address this need, we developed gene-modified natural killer (NK) cells bearing a chimeric receptor in which the activating receptor NKG2D is fused to the cytotoxic ζ-chain of the T-cell receptor (NKG2D.ζ). NKG2D.ζ–NK cells target MDSCs, which overexpress NKG2D ligands within the TME. We examined the ability of NKG2D.ζ–NK cells to eliminate MDSCs in a xenograft TME model and improve the antitumor function of tumor-directed chimeric antigen receptor (CAR)-modified T cells. We show that NKG2D.ζ–NK cells are cytotoxic against MDSCs, but spare NKG2D ligand-expressing normal tissues. NKG2D.ζ–NK cells, but not unmodified NK cells, secrete pro-inflammatory cytokines and chemokines in response to MDSCs at the tumor site and improve infiltration and antitumor activity of subsequently infused CAR-T cells, even in tumors for which an immunosuppressive TME is an impediment to treatment. Unlike endogenous NKG2D, NKG2D.ζ is not susceptible to TME-mediated down-modulation and thus maintains its function even within suppressive microenvironments. As clinical confirmation, NKG2D.ζ–NK cells generated from patients with neuroblastoma killed autologous intra-tumoral MDSCs capable of suppressing CAR-T function. A combination therapy for solid tumors that includes both NKG2D.ζ–NK cells and CAR-T cells may improve responses over therapies based on CAR-T cells alone.


T lymphocytes can be engineered to target tumor-associated antigens by forced expression of CARs (). Although successful when directed against leukemia-associated antigens such as CD19 (), CAR-T cell therapy for solid tumors has been less effective, with best responses in patients with minimal disease (). Solid tumors recruit inhibitory cells such as myeloid-derived suppressor cells (MDSCs) (). These immature myeloid cells are a component of innate immunity and strengthen the suppressive TME (). The frequency of circulating or intra-tumoral MDSCs correlates with cancer stage, disease progression, and resistance to standard chemo- and radio-therapies (). Hence, MDSCs are worth targeting in the quest to enhance CAR-T cell efficacy against solid tumors.

Natural killer (NK) cells, a lymphoid component of the innate immune system, produce MHC-unrestricted cytotoxicity and secrete pro-inflammatory cytokines and chemokines (). NK cells also modulate the activity of antigen-presenting myeloid cells within lymphoid organs, and recruit and activate effector T cells at sites of inflammation (). NK cells express NKG2D, a cytotoxicity receptor that is activated by non-classical MHC molecules expressed on cells stressed by events such as DNA damage, hypoxia, or viral infection (). NKG2D ligands are overexpressed on several solid tumors and on tumor-infiltrating MDSCs (). NK cells, therefore, could alter the TME in favor of an antitumor response by eliminating suppressive elements such as MDSCs. However, the NKG2D cytotoxic adapter molecule, DAP10, is downregulated by suppressive molecules of the TME, such as TGFβ (), limiting the antitumor functions of NK cells.

To overcome the repressive effect of the solid TME on NKG2D function, we used a retroviral vector to modify NK cells with a chimeric NKG2D receptor (NKG2D.ζ) comprising the extracellular domain of the native NKG2D molecule fused to the intracellular cytotoxic ζ-chain of the T-cell receptor (). We hypothesized that primary human NK cells expressing NKG2D.ζ (NKG2D.ζ–NK cells) would maintain NKG2D.ζ expression within the suppressive TME, kill NKG2D ligand-expressing MDSCs, secrete pro-inflammatory cytokines and chemokines, and recruit and activate effector cells, including CAR-T cells, derived from the adaptive immune system. These benefits are not attainable from NK cells expressing the native NKG2D receptor as its functions are down-modulated in the TME. Here we show that when NK cells express NKG2D.ζ, immune suppression is sufficiently countered to enable tumor-specific CAR-T cells to persist within the TME and eradicate otherwise resistant tumors.


Cytokines, cell lines, and antibodies.

Recombinant human interleukin (IL)2 was obtained from National Cancer Institute Biologic Resources Branch (Frederick, MD). Recombinant human IL6, GM-CSF, IL7, and IL15 were purchased from Peprotech (Rocky Hill, NJ, USA). The human neuroblastoma cell line LAN-1 was purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM culture medium supplemented with 2 mM L-glutamine (Gibco-BRL) and 10% FBS (Hyclone, Waltham, MA, USA). The human CML cell line K562 was purchased from American Type Culture Collection and cultured in complete-RPMI culture medium composed of RPMI-1640 medium (Hyclone) supplemented with 2 mM L-glutamine and 10% FBS. A modified version of parental K562 cells, genetically modified to express a membrane-bound version of IL15 and 41BB-ligand, K562-mb15–41BB-L, was kindly provided by Dr. Dario Campana (National University of Singapore). All cell lines were verified by either genetic or flow cytometry-based methods (LAN-1 and K562 authenticated by ATCC in 2009) and tested for mycoplasma contamination monthly via MycoAlert (Lonza) mycoplasma enzyme detection kit (last mycoplastma testing of LAN-1, K562 parental line, and K562-mb15–41BB-L on November 2, 2018; all negative). All cell lines were used within one month of thawing from early-passage (< 3 passages of original vial) lots.

CAR-encoding retroviral vectors.

The construction of the SFG-retroviral vector encoding GD2-CAR.41BB.ζ, as shown in Supplementary Fig. S1A, was previously described (). The SFG-retroviral vector encoding NKG2D.ζ, an internal ribosomal entry site (IRES), and truncated CD19 (tCD19), was generated by sub-cloning NKG2D.ζ from a retroviral vector () kindly provided by Dr. Charles L. Sentman (Dartmouth Geisel School of Medicine, Hanover, NH, USA) into pSFG.IRES.tCD19 (). RD114-speudotyped viral particles were produced by transient transfection in 293T cells, as previously described ().

Expansion and retroviral transduction of human NK and T cells.

Human NK cells were activated, transduced with retroviral constructs (Fig. 1A) and expanded as previously described by our laboratory (). Briefly, peripheral blood mononuclear cells (PBMCs) obtained from healthy donors under Baylor College of Medicine IRB-approved protocols, were cocultured with irradiated (100 Gy) K562-mb15–41BB-L at a 1:10 (NK cell:irradiated tumor cell) ratio in G-Rex® cell culture devices (Wilson Wolf, St. Paul, MN, USA) for 4 days in Stem Cell Growth Medium (CellGenix) supplemented with 10% FBS and 500 IU/mL IL2. Cell suspensions on day 4 (containing 50–70% expanded/activated NK cells) were transduced with SFG-based retroviral vectors, as previously described (). The transduced cell population was then subjected to secondary expansion to generate adequate cell numbers for experiments in G-Rex® devices at the same NK cell:irradiated tumor cell ratio with 100 IU/mL IL2. This 17-day human gene-modified NK cell protocol resulted in > 97% pure CD56+/CD3 NK cell population with avg. 77.4% ± 18.2% (n = 25) of NK cells transduced with the construct of interest. For most experiments, transduced NK cells were purified to > 95% by magnetic column selection of truncated CD19 selection marker-positive cells.

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NKG2D.ζ–NK cells expand and kill ligand-expressing targets.

(A) Schematic of SFG-based retroviral vector constructs for transduction of human NK cells. (B) Human NK cells were expanded as described in Methods and percentage of CD56+/CD3 NK cells at time of retroviral transduction (day 4) is shown. Expanded NK cells (red circle in A) purified via depletion of CD3+ cells were transduced with NKG2D.ζ retroviral vector or empty vector control (referred to as “unmodified”), and transduction efficiencies are shown inset. (C) NKG2D expression on NK cells (MFI, inset) was assessed with isotype antibody as control. Non-transduced NK cells exhibited similar NKG2D expression to empty vector-transduced NK cells. * p = 0.003 vs. unmodified condition. (D) Expression of NKG2D (absolute MFI on y-axis) on NK cells from each donor (n = 25) transduced with either empty vector or NKG2D.ζ construct was determined by flow cytometry. Each pair of data points connected by a line represent cells from a single donor, to confirm surface expression of our chimeric molecule after transduction. Black line with grey block next to each group are mean MFI ± SEM. (E) NKG2D.ζ–NK cell cytotoxicity against K562 and LAN-1 tumor targets in a 4-hour 51Cr-release assay. Given that K562 and LAN-1 are both NK-sensitive targets, low E:T ratios were utilized to observe differences. Experiment is representative of at least three separate determinations from n = 10 donors. * p < 0.01 vs. unmodified NK cells at same E:T ratio. (F) NKG2D.ζ–NK cells were expanded after transduction culture (as shown in schema), and fold-expansion and cytotoxicity both pre- (day 7) and post- (day 17) secondary expansion were determined.

For production of GD2.CAR-T cells (autologous to MDSCs and NK cells), PBMCs from healthy donors were suspended in T-cell medium (TCM) consisting of RPMI-1640 supplemented with 45% Click’s Medium (Gibco-BRL), 10% FBS, and 2 mM L-glutamine, and cultured in wells pre-coated with CD3 (OKT3, CRL-8001, American Type Culture Collection) and CD28 (Clone CD28.2, BD Biosciences) antibodies for activation. Human IL15 and IL7 were added on day +1, and cells underwent retroviral transduction on day +2, as previously described (). T-cells were used for experiments between days +9 to +14 post-transduction, with phenotype as shown in Supplementary Figs. S1BC.

Induction and enrichment of human MDSCs.

Our method for ex vivo generation of human PBMC-derived MDSCs was derived from published reports (), with slight modifications. Briefly, PBMCs were sequentially depleted of CD25hi-expressing cells and CD3-expressing cells by magnetic column separation (Miltenyi Biotec). Resultant CD25lo/−, CD3 PBMCs were plated at 4×106 cells/mL in complete-RPMI medium with human IL6 and GM-CSF (both at 20 ng/mL) onto 12-well culture plates (Sigma Corning) at 1 mL/well. Plates were incubated for 7 days with medium and cytokines being replenished on days 3 and 5. Resultant cells were harvested by gentle scraping and MDSCs were purified by magnetic selection using CD33 magnetic microbeads (Miltenyi Biotec). Cells were analyzed by multi-color flow cytometry for CD33, CD14, CD15, HLA-DR, CD11b, CD83, and CD163 (BD Biosciences). MDSCs were defined as either monocytic (M-MDSCs; CD14+, HLA-DRlow/−), PMN-MDSCs (CD14, CD15+, CD11b+), or early-stage MDSCs (lineage, HLA-DRlow/−, CD33+), as per published guidelines (). In addition to the above markers, MDSCs were stained for PD-L1, PD-L2, and NKG2D ligands via an NKG2D-Fc chimera (BD Biosciences) followed by FITC-labeled anti-Fc. This pan-ligand staining approach was determined to be the most efficient way to assess NKG2D ligand expression on human MDSCs because (1.) NKG2D ligand expression had not previously been reported for human MDSCs and thus simultaneous evaluation of the eight different NKG2D ligands would have been required, and (2.) we found poor reproducibility in staining patterns using individual commercially-available ligand antibodies, even within the same donor.

In vitro T-cell suppression assay.

T-cell proliferation was assessed using Cell Trace Violet (Thermo Fisher) dye dilution analysis, as per manufacturer’s recommendations. Briefly, 1×105 Cell Trace Violet-labeled T cells (isolated at the time of MDSC generation) were plated onto 96-well plates in the presence of plate-bound 1 μg/mL CD3 and 1 μg/mL CD28 antibodies with 50 IU/mL IL2 in the absence or presence of autologous MDSCs or peripheral blood monocytes (as a myeloid cell control) at 1:1, 4:1 and 8:1 T cell:MDSC ratios. In some experiments, only the 4:1 ratio is shown as this was determined as optimal for assessment of suppression. After 4 days of coculture, T cells were labeled with CD3 antibody and assessed for cell division using Cell Trace Violet dye dilution by flow cytometry. Percent suppression was calculated as follows: [(% proliferating T cells in the absence of MDSCs – % proliferating T cells in presence of MDSCs)/% proliferating T cells in the absence of MDSCs] x 100. Proliferation was defined as % T cells undergoing active division as represented by Cell Trace Violet dilution peaks, as previously described ().

In vitro CAR-T chemotaxis assay.

Transwell 5-μm pore inserts (Corning, Somerset, NJ) for migration experiments were prepared by coating with 0.01% gelatin at 37 °C overnight, followed by 3 μg of human fibronectin (Life Technologies, Grand Island, NY) at 37 °C for 3 hours to mimic endothelial and extracellular matrix components, as previously described (). Briefly, 2×105 purified GD2.CAR-T cells were placed in 100 μL of TCM in the upper chambers of the pre-coated Transwell inserts that were then transferred into wells of a 24-well plate. Culture supernatants (400 μL) from NKG2D.ζ or unmodified NK cells cultured with autologous MDSCs or monocytes, were placed in the lower chambers of the wells. Plain medium or medium supplemented with 1 μg/mL of the T-cell recruiting chemokine, MIG, served as negative and positive controls, respectively. The plates were then incubated for 4 hours at 37 °C with 5% CO2, followed by a 10-minute incubation at 4 °C to loosen any cells adhering to the undersides of the insert membranes. The fluid in the lower chambers was collected separately and migrated cells were counted using trypan blue exclusion. The cells were analyzed for CAR expression by flow cytometry to confirm phenotype of migrated T cells.

In vivo tumor microenvironment model.

12–16 week old female NSG mice were implanted subcutaneously in the dorsal right flank with 1×106 Firefly luciferase(FfLuc)-expressing LAN-1 neuroblastoma cells admixed with 3×105 ex vivo-generated MDSCs, suspended in 100 μL of basement membrane Matrigel (Corning). Matrigel basement membrane was important in keeping tumor and MDSCs confined so as to establish a localized solid TME. 10–14 days later, when tumors measured at least 100 mm3 by caliper measurement, mice were injected intravenously with 5×106 GD2.CAR-T cells. Tumor growth was measured twice weekly by live bioluminescence imaging using the IVIS® system (IVIS, Xenogen Corporation) 10 minutes after 150 mg/kg D-luciferin (Xenogen)/mouse was injected intraperitoneally. In experiments examining the ability of NKG2D.ζ–NK cells to reduce intra-tumoral MDSCs, 1×107 unmodified or NKG2D.ζ–NK cells were injected intravenously when tumors measured at least 100 mm3. At end of experiment, tumors were harvested en bloc, digested ex vivo, and intra-tumoral human MDSCs (CD33+, HLA-DRlow cells) were enumerated by flow cytometry. The absolute number of human MDSCs within a tumor digest was enumerated per mouse (n = 5 mice/group), compared to pre-treatment MDSC numbers, and presented as mean % MDSCs remaining per treatment group. In experiments examining the effects of NKG2D.ζ–NK cells on GD2.CAR-T cell antitumor activity, 5×106 (cell dose chosen to mitigate direct antitumor effects of NK cells) unmodified or NKG2D.ζ–NK cells were injected intravenously 3 days prior to GD2.CAR-T injection. In GD2.CAR-T cell homing experiments, CAR-T were transduced with GFP-luciferase retroviral construct prior to injection into mice bearing unmodified tumor cells (). Mice received 5000 IU human IL2 intraperitoneally three times per week for 3 weeks following NK cell injection to promote NK cell survival in NSG mice (). Tumor size was measured twice weekly with calipers and the mice were imaged for bioluminescence signal from T cells at the same time. Mice were euthanized for excessive tumor burden, as per protocol guidelines. The animal studies protocol was approved by Baylor College of Medicine Institutional Animal Care and Use Committee and mice were treated in strict accordance with the institutional guidelines for animal care.

Immunohistochemistry of neuroblastoma xenografts.

On day 32 of in vivo experiments, animals were sacrificed, tumors were harvested and sectioned bluntly ex vivo to separate tumor periphery (outer 1/3 of tumor volume) vs. core (non-necrotic inner 2/3 of tumor volume), and n = 5 sections/tumor sample were analyzed for presence of GD2.CAR-T and NKG2D.ζ–NK cells by H&E and human CD3 and CD57 immunostaining performed by the Human Tissue Acquisition and Pathology Core of Baylor College of Medicine. Lack of CD57 expression on infused GD2.CAR-T was confirmed by flow cytometry prior to administration. CD57 was chosen as the marker for NK cells in tumor tissue in our study because LAN-1 tumors naturally express the prototypical NK marker CD56, truncated CD19 expression was inadequate for in situ staining, and CD57 had previously been used as a marker for tissue-localized activated NK cells (). The number of human CD3+ and CD57+ cells in representative sections of tumors from periphery vs. core of the treatment groups indicated were enumerated per high-powered field (HPF) at 40x magnification and percent of the total number of cells enumerated within tumors found in the periphery vs. core in each treatment group indicated from tumors with and without MDSCs is shown as mean ± SEM of n = 5 sections/periphery or core, n = 5 tumors/group.

Analysis of intra-tumoral MDSCs from patients with neuroblastoma.

Tumor tissue and matched peripheral blood of neuroblastoma patients obtained in the context of a specimen/laboratory study after patient identification had been removed were thawed and analyzed for MDSC subsets by flow cytometry or utilized in in vitro assays, as described in legends or Results. The tissue acquisition protocol was performed after review and approval by the Baylor College of Medicine Institutional Review Board. Briefly, subjects with a diagnosis of high-risk or intermediate-risk neuroblastoma were eligible to participate. Written informed consent, or appropriate assent for participation, in accordance with the Declaration of Helsinki was obtained from each subject or subject’s guardian for procurement of patient blood and tumor tissue and for subsequent analyses of stored patient materials.


Data are presented as mean ± SEM of either experimental replicates or number of donors, as indicated. Paired two-tailed t-test was used to determine significance of differences between means with p < 0.05 indicating a significant difference. For in vivo bioluminescence, changes in tumor radiance from baseline at each time point were calculated and compared between groups using two-sample t-test. Multiple group comparisons were conducted via ANOVA via GraphPad Prism v7 software. Survival determined from the time of tumor cell injection was analyzed by Kaplan-Meier and differences in survival between groups were compared by the log-rank test.


NKG2D.ζ NK cells expand and have cytotoxicity against target cells.

To increase killing of NKG2D ligand-expressing MDSCs, we generated primary human NK cells stably expressing NKG2D.ζ and a truncated CD19 (tCD19) marker from a retroviral vector (Fig. 1A). NK cells were expanded from PBMCs obtained from normal donors, transduced with retroviral construct expressing chimeric NKG2D, then cultured for 3 additional days. Transduction efficiency, as measured by the expression of tCD19 on CD56+CD3 NK cells after the additional 3 days, was 71.3 ± 16% (n = 25 normal donors) and produced a 5.4 ± 1.1-fold increase in NKG2D expression on the NK cell surface (Fig. 1BD). NKG2D.ζ–NK cells showed greater cytotoxicity (79.2 ± 5.6%, n = 10 normal donors) against wild-type K562, a highly NK cell-sensitive tumor cell line that naturally expresses NKG2D ligands, than mock vector-transduced (hereafter referred to as, unmodified) NK cells (40.5 ± 2.1%) at 2:1 E:T ratio in a 4-hr cytotoxicity assay (Fig. 1E). In contrast, transgenic NKG2D.ζ expression did not increase NK cell killing of LAN-1 neuroblastoma cells that are marginally NK-sensitive, but lack NKG2D ligands. To determine if in vitro expansion affected the cytotoxic function of NKG2D.ζ–NK cells, we secondarily expanded NKG2D.ζ–NK cells for an additional 10-days (Fig. 1F schema). As seen in Fig. 1F, NKG2D.ζ–NK cells expanded (120 ± 7.3-fold by day 17 of culture; n = 10 donors) similarly to unmodified and non-transduced NK cells and maintained stable cytotoxic function between days 7 and 17 of expansion. Thus, we generated and expanded high numbers of primary human NKG2D.ζ-expressing NK cells capable of cytotoxicity against ligand-expressing targets, even after prolonged culture.

Transgenic NKG2D.ζ is unaffected by TGFβ or soluble NKG2D ligands.

Expression of the native NKG2D receptor on NK cells is down-modulated by tumor-derived TGFβ and soluble NKG2D ligands, both of which are abundant in the TME () and likely impair NK cell function in solid tumors. To determine the effect of TGFβ and soluble NKG2D ligands on NKG2D.ζ receptor expression and function, we cultured NKG2D.ζ–NK cells in the presence of TGFβ or the soluble NKG2D ligands, MICA and MICB, and examined NKG2D expression and NK cytotoxicity after 24-, 48-, and 72-hours. After exposure to TGFβ or soluble MICA/B, unmodified NK cells significantly down-regulated NKG2D (MFI of 25 vs. 95 in non-exposed NK cells at 48 hours) and were less cytotoxic (20 ± 5.1% killing vs. 40 ± 3.7% killing by non-exposed NK cells at 48 hours) to NKG2D ligand-expressing K562 targets (Fig. 2A,B).B). In contrast, NKG2D.ζ–NK cells maintained NKG2D expression and cytotoxicity after exposure to the same concentrations of TGFβ and soluble MICA/B (Fig. 2C,D).D). This lack of sensitivity to down-regulation by these tumor-associated components should benefit the function of NKG2D.ζ–NK cells within the TME.

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Transgenic NKG2D.ζ is unaffected by TGFβ or soluble NKG2D ligands.

NKG2D.ζ or unmodified NK cells (n = 5 donors) were cultured in the presence of TGFβ (5 ng/mL) (A, B) or the soluble NKG2D ligands MICA and MICB (C, D) for 24-, 48-, and 72-hours. NKG2D receptor expression was determined by flow cytometry and NK cytotoxicity against K562 targets was assessed in a 4-hr Cr-release assay at an 5:1 E:T ratio using 48-hr exposed NK cells. Viability of transduced NK cells after exposure to TGFβ for 24, 48, and 72 hours, as assessed by 7-AAD vital staining, was > 90%. * p = 0.001 vs. non-TGFβ/MICA-treated NK groups at same time-points.

Human MDSCs express NKG2D ligands and are killed by NKG2D.ζ–NK cells.

To study the effects of human NK cells on autologous MDSCs, we generated human MDSCs by culture of CD3-/CD25lo PBMC with IL6 plus GM-CSF for 7 days, followed by CD33+ selection, as described in the Methods. The phenotypic characterization of these MDSCs and confirmation of their suppressive capacity is shown in Supplementary Fig. S2. Routinely, our ex vivo-generated MDSCs contained monocytic (M)-MDSC and early(e)-MDSC subsets, with few (avg. < 1%) polymorphonuclear (PMN)-MDSCs (Supplementary Fig. S2A), roughly reflecting the subset composition reported in patients with solid tumors (). The MDSCs expressed the suppressive factors TGFβ, IL6, IL10, and PDL-1 in amounts often greater than tumor cells (Supplementary Figs. S2BC), and suppressed proliferation and cytokine secretion by autologous T cells stimulated with plate-bound CD3/CD28 antibodies (Supplementary Figs. S2DE) and by 2nd generation GD2.CAR-T cells encoding 4–1BB and CD3-ζ endodomains stimulated with the GD2+ tumor line LAN-1 (Supplementary Figs. S2FG). As seen in Fig. 3A, MDSCs expressed as much or more NKG2D ligand than the positive control tumor line, K562 (ligand MFI of 78.2 vs. 29.7, respectively). Freshly isolated peripheral blood T cells did not express NKG2D ligands, whereas immature and mature dendritic cells expressed little, consistent with previous data (). The neuroblastoma cell line, LAN-1, subsequently used in our in vivo TME model, did not express NKG2D ligands.

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Human MDSCs express ligands for NKG2D and are killed by NKG2D.ζ–NK cells.

(A) NKG2D ligand expression on human MDSCs by flow cytometry. Immature dendritic cells (iDC) and mature DCs (mDC) were used as myeloid controls. T cells activated with CD3 and CD28 mAbs plus 100 IU/mL IL2 for 24 hours were used as lymphocyte control. LAN-1 and K562 cells were used as negative and positive controls, respectively. MFI of NKG2D ligand expression in parenthesis. Representative data from single donor (of n = 25 normal donors). Isotype control for NKG2D staining routinely fell within the 1st log. (B) NKG2D.ζ–NK cell cytotoxicity against autologous MDSCs as targets in a 4-hour 51Cr-release assay. In some wells of the cytotoxicity assay, a blocking mAb to NKG2D was added. Representative data from triplicate samples per data point from a single donor (of n = 25 normal donors) is shown. * p < 0.01 vs. unmodified NK cells at same E:T ratio. (C) In the same experiment as (B), the same batch of NKG2D.ζ–NK cells were analyzed for cytotoxicity against autologous B cells, monocytes, monocyte-derived iDC and mDC, and activated T cells (n = 10 donors examined). (D) M-MDSC frequency by flow cytometry from neuroblastoma tumor samples obtained from high-risk patients, as described in Methods. (E) Cytotoxicity by NKG2D.ζ–NK cells derived from patient PBMC (harvested and frozen at time of tumor sampling) against autologous tumor-derived MDSCs in a 4-hour 51Cr-release assay. Data shown are from triplicate samples per data point at a 10:1 E:T ratio. * p < 0.001 vs. unmodified NK cells from same donor. (F) NKG2D.ζ–NK cells were cocultured with autologous MDSCs at 1:1 ratio plus low-dose 50 IU/mL IL2 to maintain NK survival, and fold change in the number of each cell type from the start of coculture was determined by flow cytometry at indicated time-points. * p < 0.001 vs. NK/MDSC fold-change in unmodified NK cell cocultures. (G) Cell-free supernatants were harvested from cocultures at day 3 and analyzed for IFNγ, TNFα, IL6, and IL10 by ELISA. # p < 0.01 vs. corresponding cytokine in cocultures with unmodified NK cells. (H) NKG2D ligand expression was determined for activated T cells (ATCs) expressing NKG2D.ζ and NKG2D.ζ–NK cells. Expression of NKG2D ligands on non-transduced ATCs as control for T-cell activation. (I) NKG2D.ζ–NK cells or NKG2D.ζ T cells were cocultured with autologous ATCs at 1:1 ratio and fold change in the number of each cell type from the start of coculture was determined by flow cytometry at indicated time-points. * p < 0.001 vs. ATC fold-change at days 0 and 3 cocultures.

To evaluate MDSC susceptibility to killing by NKG2D.ζ–NK cells, we performed both short- and long-term killing assays. Fig. 3B shows enhanced killing of MDSCs by autologous NKG2D.ζ–NK cells compared to unmodified NK cells (35 ± 5.5% vs. 8 ± 2.4% cytotoxicity, respectively, at an E:T ratio of 5:1) in a 4-hr chromium-release assay. MDSC killing was dependent on NKG2D, as pre-incubation with an NKG2D blocking Ab reduced the cytotoxicity to levels achieved by unmodified NK cells. NKG2D.ζ–NK cells mediated no cytotoxicity against other autologous immune cells such as freshly-isolated monocytes, monocyte-derived mature dendritic cells, T cells, or B cells (Fig. 3C). Only immature dendritic cells, which expressed little NKG2D ligand (approx. 7% of cells; MFI 11.4), were mildly susceptible to lysis by NKG2D.ζ–NK cells (4.2 ± 1.7 % lysis at an E:T ratio of 20:1). As confirmation of the clinical applicability of our approach, we assessed whether NKG2D.ζ–NK cells generated from patient PBMCs were able to kill highly suppressive MDSCs isolated from the patient’s tumor. Tumor samples obtained from two patients with high-risk neuroblastoma at time of first biopsy/resection contained M-MDSCs (Fig. 3D). NKG2D.ζ–NK cells generated from patient PBMCs (harvested and frozen at time of tumor sampling) mediated significant cytotoxicity in vitro against M-MDSCs purified from patient tumors, whereas unmodified patient NK cells did not (Fig. 3E). These results provide further clinical evidence for the capacity of NKG2D.ζ–NK cells to eliminate MDSCs in patients with suppressive TMEs.

To determine whether NKG2D.ζ–NK cells could control MDSC survival in long-term cultures, we cocultured NKG2D.ζ–NK cells with autologous MDSCs at a 1:1 ratio for 7 days in the presence of low-dose IL2 to maintain NK survival, and quantified each cell type by flow cytometry every two days. As shown in Fig. 3F, NKG2D.ζ–NK cells expanded in cocultures (mean 9.5 ± 0.7-fold increase) with a concomitant reduction in MDSCs (mean 81.3 ± 9.4-fold decrease), whereas unmodified NK cells failed to expand or eliminate MDSCs. NK cells cultured alone or with autologous monocyte controls did not expand (0.8 ± 0.1-fold change). As seen in Fig. 3G, NK cell expansion and MDSC reduction correlated with a shift in the culture cytokine milieu from one that is immune suppressive (more IL6 and IL10; less IFN-γ and TNF-α) in cocultures containing unmodified NK cells, to one that is immune stimulatory and enhances CAR-T antitumor function (less IL6 and IL10; more IFN-γ and TNF-α) in cocultures containing NKG2D.ζ–NK cells. Hence, NKG2D.ζ–NK cells mediate potent cytotoxicity against suppressive MDSCs via their highly expressed NKG2D ligands. In addition, through selective depletion of MDSCs in combination with immune stimulatory cytokine secretion, NKG2D.ζ–NK cells skew the cytokine microenvironment to one that can support CAR-T effector functions ().

Previous studies have reported that expression of chimeric NKG2D constructs in T lymphocytes can direct these cells to target NKG2D ligand-expressing tumors (). However, activated T cells (ATCs) themselves upregulate NKG2D ligands (), with variable ligand expression intensity dependent on the T-cell activation protocol employed, leading to fratricide when the chimeric NKG2D is expressed. To determine if this off-tumor side-effect occurred when the same NKG2D.ζ was expressed in NK cells, we compared the killing of ATCs by autologous NK cells or by autologous T cells expressing our NKG2D.ζ transgene. ATCs and NKG2Dζ.-T cells both upregulated NKG2D ligands during ex vivo expansion with CD3/CD28 antibodies plus IL7 and IL15, whereas NKG2D.ζ-transduced NK cells undergoing expansion in our K562-mb15–41BB-L culture system did not (Fig. 3H). Coculture without additional stimulation of NKG2D.ζ-T cells with autologous ATCs produced fratricide, of both the NKG2D.ζ effector T cells (35 ± 7.2% decrease in cell number) and the non-transduced ATC targets (98 ± 11.5% decrease in cell number) (n = 3). By contrast, ATC numbers were unaffected by coculture with autologous NKG2D.ζ–NK cells (Fig. 3I). These results show that NK cells expressing NKG2D.ζ can kill autologous MDSCs while sparing other NKG2D ligand expressing populations, thus avoiding the fratricide seen with NKG2D.ζ-expressing T cells.

NKG2D.ζ–NK cells eliminate intra-tumoral MDSCs and reduce tumor burden.

To determine if NKG2D.ζ–NK cells could eliminate MDSCs from tumor sites in vivo, we created an MDSC-containing TME in a xenograft model of neuroblastoma. We chose NKG2D ligand-negative LAN-1 tumor for this experiment so that the effects of NKG2D.ζ–NK cells on MDSCs were not confused with their effects on the tumor cells. LAN-1 tumor cells admixed with human MDSCs were inoculated subcutaneously in NSG mice. These animals had increases in the suppressive cytokines IL10 (10-fold vs. tumor alone) and TGFβ (2.6-fold vs. tumor alone) in circulation by day 16 as compared to animals bearing tumors initiated without MDSCs, and the resultant tumors grew more rapidly due to increased neovascularization and tumor-associated stroma (Supplementary Fig. S3AD), consistent with clinical reports of MDSC-dense tumors (). As seen in Fig. 4A, in mice bearing NKG2D ligand-negative tumors without MDSCs, a single infusion of 1×107 NKG2D.ζ–NK cells resulted in a small delay in tumor growth but eventual progression, suggesting that the LAN-1 tumor itself (a marginally NK-sensitive target) can be killed at higher NK cell doses independent of NKG2D ligand expression. In mice bearing MDSC-containing tumors, 1×107 NKG2D.ζ–NK cells inhibited tumor growth (Fig. 4B), reduced NKG2D ligand-expressing intra-tumoral MDSCs with only 8.7 ± 3.5% of the input MDSCs remaining (Fig. 4C), and prolonged mouse survival (median survival of 73 days vs. 29 days after unmodified NK cells; Fig. 4D). Since LAN-1 tumor cells do not express NKG2D ligands and are only marginally sensitive to ligand-independent lysis, tumors subsequently regrew in these mice once the NKG2D.ζ–NK cells had disappeared (> day 40). Thus, NKG2D.ζ–NK cells can traffic to tumor sites and reduce intra-tumoral MDSCs but cannot themselves eradicate NKG2D ligand-negative malignant cells in our model.

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NKG2D.ζ–NK cells eliminate intra-tumoral MDSCs and reduce tumor burden.

LAN-1 tumor cells, either alone (A) or admixed with human MDSCs (B), were injected S.C. in the flanks of NSG mice. When tumors reached a volume of approx. 100 mm3 (day 14, gray block arrow inset), no NK cells (PBS control), 1×107 unmodified or NKG2D.ζ–NK cells were injected I.V. and tumor growth was measured over time via calipers. * p < 0.03 vs. other conditions shown at same time point. (C) On day 26, intra-tumoral human MDSCs (CD33+, HLA-DRlow) were enumerated by flow cytometry and are presented as mean % MDSCs remaining per treatment group. ** p < 0.005 vs. unmodified NK treatment. (D) Survival of groups by Kaplan-Meyer analysis. # p = 0.024. Representative experiment of three performed.

NKG2D.ζ–NK cells secrete chemokines that recruit GD2.CAR-T cells.

To determine if NKG2D.ζ–NK cells can recruit T cells modified with a tumor-specific CAR to tumor sites containing MDSCs, we cocultured NKG2D.ζ–NK cells with autologous MDSCs and analyzed culture supernatants for chemokines by multiplex ELISA. Compared to unmodified NK cells, NKG2D.ζ–NK cells produce significantly greater CCL5 (RANTES; 10-fold increase), CCL3 (MIP-1α; 2-fold increase), and CCL22 (MDC; 5-fold increase) in response to autologous MDSCs (Fig. 5A). Large amounts of CXCL8 (IL8) were also produced, but there was no significant difference from the production by unmodified NK cells. Analysis of chemokine receptor expression on 2nd generation GD2.CAR-T cells revealed CXCR1 (binds CXCL8), CCR2 (binds CCL2), CCR5 (binds CCL3), and CCR4 (binds CCL5) (see Supplementary Fig. S1C). These GD2.CAR-T cells were assayed for chemotaxis to supernatants derived from unmodified or NKG2D.ζ–NK cells cocultured with autologous MDSCs. Supernatants from NKG2D.ζ–NK cell-containing cocultures induced chemotaxis of 41.1 ± 5.5% of GD2.CAR-T cells (Fig. 5B), whereas supernatants from unmodified NK cells induced chemotaxis no greater than produced by medium (14.9 ± 6.4% vs. 17.3 ± 1.9%, respectively). Chemotaxis was not induced by supernatants from unmodified or NKG2D.ζ–NK cells cocultured with monocytes. Thus, following their encounter with MDSCs, NKG2D.ζ–NK cells secrete chemokines that recruit CAR-Ts in vitro.

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NKG2D.ζ–NK cells secrete chemokines that recruit GD2.CAR-T cells.

(A) NKG2D.ζ or unmodified NK cells were cocultured with autologous MDSCs and cell-free culture supernatants harvested at 48 hours were analyzed for chemokines CXCL8, CCL5, CCL3, and CCL22 by ELISA. Shown are mean chemokine concentration ± SEM for n = 5 cocultures/donor (data from one of five representative donors is shown). * p < 0.005 vs. unmodified NK cocultures. (B) GD2.CAR-T cells were assayed for chemotaxis in Transwells (described in Methods) in response to supernatants derived from unmodified or NKG2D.ζ–NK cells cocultured with autologous MDSCs. Supernatants derived from monocyte (non-suppressive myeloid cell control)-stimulated NK cells were also used. # p < 0.001 vs. Medium, ** p = 0.009 vs. “unmodified NK plus MDSCs” condition. (C) LAN-1 tumor cells, alone or admixed with human MDSCs, were injected S.C. into the flank of NSG mice. When tumors reached a volume ~100 mm3, 5×106 GD2.CAR-T cells were injected I.V. alone on day 13 (GD2.CAR-T), or preceded by 5×106 NKG2D.ζ–NK cells I.V. injected on day 10 (chNK + GD2.CAR-T). GD2.CAR-T signal at tumor site was measured over time via live-animal bioluminescence imaging. (D) Shown is mean ± SEM (n=5 mice/group) bioluminescent signal of GD2.CAR-T cells expressed as radiance. * p = 0.01 vs. all other groups.

NKG2D.ζ NK cells improve GD2.CAR-T cell trafficking to tumor sites.

To determine the effects of the MDSC-induced, NKG2D.ζ–NK cell chemokines on CAR-T cell recruitment in vivo, we used our MDSC-containing TME xenograft model (see Fig. 4). When tumors reached a volume of ~100 mm3 (day 10), 5×106 NKG2D.ζ–NK cells were infused, followed three days later (day 13) by infusion of 5×106 luciferase gene-transduced GD2.CAR-T cells. Tumor localization and expansion of GD2.CAR-T cells was measured over time via live-animal bioluminescence imaging. As seen in Fig. 5C, GD2.CAR-T cells injected alone on day 13 after tumor inoculation (without pre-administration of NKG2D.ζ–NK cells) into mice bearing tumors devoid of MDSCs localized effectively to subcutaneous tumors in the flank (4 of 5 mice showed bioluminescent signal on days 14 and 18; Fig. 5C). There was a 10.5 ± 0.8-fold increase in bioluminescent signal on day 18, with CAR-T cell bioluminescence remaining above baseline levels for the duration of the experiment (Fig. 5D). However, in tumors containing MDSCs, CAR-T cells localized poorly: only 1 of 5 mice exhibited bioluminescent signal (Fig. 5C), with only a 1.02 ± 0.1-fold increase in bioluminescent signal on day 18 and bioluminescence falling below pre-infusion levels within 10 days after injection (Fig. 5D). In contrast, pre-administration of NKG2D.ζ–NK cells on day 10 into mice bearing MDSC-containing tumors allowed subsequently infused GD2.CAR-T cells to localize effectively to tumor sites, with bioluminescence in 5 of 5 mice at the tumor site and a 10.9 ± 0.2-fold increase in bioluminescent signal on day 18, within 5 days of injection (Fig. 5D).

To determine if NKG2D.ζ–NK cells could promote GD2.CAR-T infiltration into the tumor bed, we compared the frequency of human GD2.CAR-T and human NK cells in the tumor periphery and the tumor core by immunohistochemistry (Supplementary Fig. S4AB). In tumors without MDSCs, 89 ± 11% of the total T cells in the tumor had infiltrated into the tumor core. In contrast, a much smaller fraction (39 ± 16%) infiltrated into the core of tumors containing MDSCs, suggesting TME suppression of CAR-T infiltration. However, pre-treatment of tumors containing MDSCs with NKG2D.ζ–NK cells increased the fraction of intra-tumoral CAR-T cells (70 ± 13%) within the tumor core. Equal numbers of NKG2D.ζ–NK cells were observed within both peripheral and core samples from MDSC-positive and MDSC-negative tumors (Supplementary Fig. S5), suggesting the ability of NK cells to traffic well within tumors despite the presence of MDSCs.

Elimination of MDSCs increases antitumor activity of GD2.CAR-T cells.

To determine if the activities of NKG2D.ζ–NK cells described above enhance the antitumor function of CAR-T cells, we treated mice bearing subcutaneous, luciferase-labeled neuroblastoma containing MDSCs with GD2.CAR-T cells preceded by NKG2D.ζ–NK cells, in a similar set-up to experiments in Fig. 5C. As seen in Fig. 6AB, a single injection of 5×106 NKG2D.ζ–NK cells (a dose that achieved intra-tumoral MDSC depletion with only 26.8 ± 5.8% of the input MDSCs remaining) resulted in no significant tumor regression or prolongation of survival in mice bearing xenografts containing human MDSCs. A single infusion of 5×106 GD2.CAR-T cells significantly reduced tumor in mice whose xenografts lacked human MDSCs with a median survival of 95 days (Fig. 6CD). However, the same GD2.CAR-T cells were ineffective against xenografts containing human MDSCs, worsening overall median survival to 39 days (Fig. 6B). In contrast, when the same GD2.CAR-T cell injection was preceded 3 days earlier by a single injection of 5×106 NKG2D.ζ–NK cells (that had no direct antitumor effect by themselves within the other arm of the same experiment, see Fig. 6AB), the antitumor activity of the GD2.CAR-T cells in mice bearing MDSC-containing tumors was restored to the level observed in mice whose tumors lacked MDSCs (Fig. 6C). NKG2D.ζ–NK cells pre-injection also improved the overall survival of the mice with MDSC-containing tumors to a median 120 days with durable cure in 2 of 5 mice (Fig. 6D). Taken together, our results suggest that NKG2D.ζ–NK cells not only eliminate MDSCs from the TME, but also recruit CAR-T cells to intra-tumoral sites which facilitates antitumor efficacy.

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Elimination of MDSCs by NKG2D.ζ–NK cells increases antitumor activity of GD2.CAR-T cells.

Luciferase gene-transduced LAN-1 tumor cells, alone or admixed with human MDSCs, were injected S.C. into NSG mice. (A) When tumors reached a volume ~100 mm3, no treatment (No Tx; PBS control) or 5×106 NKG2D.ζ–NK cells alone (chNK) were injected I.V. on day 10 and tumor growth was measured over time via live-animal bioluminescence imaging. Shown is mean ± SEM (n=5 mice/group) bioluminescent signal expressed as radiance. # ns, p = 0.18 vs. No treatment (+MDSC) group. (B) Survival of groups in A was determined by Kaplan-Meyer analysis. # ns, p = 0.059. (C) In other groups of mice within the same experiment, 5×106 GD2.CAR-T cells were injected I.V. alone on day 13 (GD2.CAR-T), or preceded by 5×106 NKG2D.ζ–NK cells injected on day 10 (chNK+GD2.CAR-T). * p = 0.001; # ns, p = 0.59 vs. each other. (D) Survival of groups in C by Kaplan-Meyer analysis. Representative experiment of 5 separate experiments. * p = 0.002; ** p = 0.001.


We have developed a TME-disrupting approach that eliminates MDSCs and rescues MDSC-mediated impairment of tumor-directed CAR-T cells. We show that when co-implanted with a neuroblastoma cell line, human MDSCs both enhance tumor growth and suppress the infiltration, expansion, and antitumor efficacy of tumor-specific CAR T-cells. In this model, NK cells bearing a chimeric version of the activating receptor NKG2D (NKG2D.ζ–NK cells) are directly cytotoxic to autologous MDSCs, thus eliminating MDSCs from tumors. In addition, NKG2D.ζ–NK cells secrete pro-inflammatory cytokines and chemokines in response to MDSCs at the tumor site, improving CAR-T cell infiltration and function, and resulting in tumor regression and prolonged survival compared to treatment with CAR-T cells alone. Our cell therapy approach utilizes an engineered innate immune effector that targets the TME, and shows potential to enhance efficacy of combination immune-based therapies for solid tumors.

NKG2D.ζ–NK cells directly killed highly suppressive MDSCs generated in vitro as well as those from patient tumors. NKG2D.ζ–NK cells also secreted cytokines that favored immune activation in response to MDSCs. Unmodified NK cells were unable to mediate these effects. The ability of NKG2D.ζ–NK cells to eliminate MDSCs from the TME should have several beneficial effects for antitumor immunity. First, as MDSCs express suppressive cytokines such as TGFβ and the checkpoint ligands PDL-1 and PDL-2, elimination of MDSCs should help relieve the suppression of endogenous T cell responses and potentiate the activity of adoptive T cell therapies. Given that high baseline numbers of MDSCs have been reported as a biomarker of poor response in the context of trials with the checkpoint inhibitors ipilimumab and pembrolizumab (), elimination of MDSCs by NKG2D.ζ–NK cells may also enhance checkpoint inhibition. Second, elimination of MDSCs should also decrease other MDSC-associated effects, including neovascularization via their expression of VEGF, production of immunosuppressive metabolic products such as PGE2 and adenosine, and establishment of tumor-supportive stroma via their expression of iNOS, FGF, and matrix metalloproteinases (). In short, the ability of NKG2D.ζ–NK cells to eliminate MDSCs alters the tumor microenvironment in multiple ways that should improve antitumor immunity.

Previous strategies for modulation of MDSCs within the TME have included use of agents that block single functions such as secretion of nitric oxide () or expression of checkpoint molecules (); induce MDSC differentiation such as with all-trans retinoic acid (); or eliminate MDSCs such as with the cytotoxic agents doxorubicin or cyclophosphamide (). The MDSC eliminating effects were dependent on continued administration of the agents, with a rapid rebound in MDSCs after discontinuation. Moreover, many of these agents have off-target toxicities that include damage to endogenous tumor-specific T cells. In contrast, NKG2D.ζ–NK cells produce prolonged and specific elimination of MDSCs with the potential to kill MDSCs that are recruited to the tumor from the bone marrow, while continually secreting cytokines and chemokines which respectively alter TME suppression and recruit and activate tumor-specific T cells. Thus, NKG2D.ζ–NK cells exert a prolonged combination of simultaneous immune modulatory effects that enhance antitumor immune function in ways that could not be achieved by previous methods that target MDSCs.

We observed no toxicity against normal hematopoietic cells when NKG2D.ζ was expressed in autologous human NK cells. Previous studies overexpressing an NKG2D.ζ receptor containing co-stimulatory endodomains (e.g., CD28 or 41BB) and DAP10, a signaling adaptor molecule for enhanced surface expression of NKG2D, in T cells showed activity against NKG2D ligand-overexpressing tumors, but at the cost of fratricide in vitro and lethal toxicity in mice (). Using our standard T-cell activation/expansion protocol (), we also observed upregulation of NKG2D ligands, leading to fratricide in T cells expressing NKG2D.ζ. When NKG2D.ζ-T cells engage NKG2D ligands expressed on normal tissues, they will not receive the physiologic NK cell-directed inhibitory inputs that would safely regulate this potent and unopposed chimeric receptor activity. By contrast, when NKG2D.ζ is expressed on NK cells, they are able to recognize inhibitory NK cell ligands such as self-MHC expressed on healthy self-tissues, counteracting otherwise unopposed positive signals from NKG2D ligands. Thus, an NK cell platform for NKG2D enhancement may limit toxicity while taking advantage of the cytotoxic and immune modulatory potential of the receptor-ligand system.

Unlike wild-type NKG2D, transgenic NKG2D.ζ expression and activity were not sensitive to down-modulation by TGFβ or soluble NKG2D ligands, allowing improved function in the TME. Native NKG2D relies solely on the intra-cytoplasmic adaptor DAP10 for mediating its cytolytic activity in human NK cells (). TGFβ1 and soluble NKG2D ligands both decrease DAP10 gene transcription and protein activity, and thus reduce NKG2D function in endogenous NK cells (). In contrast, transgenic NKG2D.ζ does not rely on DAP10-based signaling for its activity, since signaling occurs through the ζ-chain. Thus, this construct provides a stable cytolytic pathway capable of circumventing TME-mediated down-modulation of native NKG2D activity. A previous study expressing a chimeric NKG2D.ζ molecule that incorporated DAP10 reported enhanced NK cytotoxicity compared to NKG2D.ζ alone in vitro against a variety of human cancer cell lines as well as in a xenograft model of osteosarcoma (). However, this report did not address the susceptibility of this complex to down-modulation by TGFβ or soluble NKG2D ligands, or whether these NK cells had activity against MDSCs.

NKG2D.ζ–NK cells countered immunosuppression mediated by MDSCs leading to enhanced CAR-T cell tumor infiltration and expansion at tumor sites, CAR-T functions that are impaired in patients with solid tumors (). Unlike the GD2.CAR-T cells in our model, NKG2D.ζ–NK cells homed effectively to MDSC-engrafted tumors and released an array of chemokines that increased T cell infiltration of tumor. Unlike pharmacologic strategies aimed at enhancing leukocyte trafficking, including administration of lymphotactin or TNFα (), our approach does not require continuous cytokine administration. In fact, the ability of chimeric NKG2D to augment NK immune function specifically within the immunosuppressive TME provides for the local release of chemotactic factors, reflecting a more homeostatic method by which to increase CAR-T infiltration. Once there, CAR-T cells should meet an environment favorably modified by NKG2D.ζ–NK cell mediated elimination of MDSCs and production of pro-inflammatory cytokines. Indeed, elimination of MDSCs from a GD2+ tumor xenograft enhanced the activity of GD2.CAR-T cells in our model, including T-cell survival and intratumoral expansion. Given the suppressive effects of MDSCs in neuroblastoma (), the model shows how reversal of an MDSC-mediated suppressive microenvironment can improve antitumor functions of effector T cells.

Clinical neuroblastoma contains intense infiltrates of MDSCs (), which are not included in tumor xenograft models currently used to study human cell therapeutics. Our data suggest that co-inoculation of tumors with suppressive components (such as MDSCs) can model TME-mediated suppression of CAR-T activity against solid tumors, and provides a method by which to understand and counter immunosuppression. Although NSG mice lack a complete immune system in which to examine the effects of multiple endogenous immune components, our ability to engraft exogenous components (e.g., human MDSCs) within our TME model provides the possibility of simulating different immunosuppressive aspects of the solid TME. In fact, further model development utilizing human inhibitory macrophages and regulatory T cells (Tregs) as additional suppressive components of the TME is currently underway in our laboratory.

In summary, we describe an approach to reverse the suppressive TME using engineered human NK cells. We have shown that generation and expansion of our NK cell product is feasible and that NKG2D.ζ–NK cells have antitumor activity within a suppressive solid tumor microenvironment without toxicity to normal NKG2D ligand-expressing tissues. Hence, the elimination of suppressive MDSCs by NKG2D.ζ–NK cells may safely enhance adoptive cellular immunotherapy for neuroblastoma and for many other tumors that are supported and protected by MDSCs.

Other posts on this site on Immunotherapy and Cancer include

Combined anti-CTLA4 and anti-PD1 immunotherapy shows promising results against advanced melanoma

Molecular Profiling in Cancer Immunotherapy: Debraj GuhaThakurta, PhD

Pancreatic Cancer: Genetics, Genomics and Immunotherapy

$20 million Novartis deal with ‘University of Pennsylvania’ to develop Ultra-Personalized Cancer Immunotherapy

Upcoming Meetings on Cancer Immunogenetics

Tang Prize for 2014: Immunity and Cancer

ipilimumab, a Drug that blocks CTLA-4 Freeing T cells to Attack Tumors @DM Anderson Cancer Center

Juno’s approach eradicated cancer cells in 10 of 12 leukemia patients, indicating potential to transform the standard of care in oncology

Report on Cancer Immunotherapy Market & Clinical Pipeline Insight

New Immunotherapy Could Fight a Range of Cancers

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Cell Research News – What’s to Follow?

Larry H. Bernstein, MD, FCAP, Reporter

Leaders in Pharmaceutical Intelligence



Stem Cell Research ‘Holy Grail’ Uncovered, Thanks to Zebrafish

By Estel Grace Masangkay

With help from the zebrafish, a team of Australian researchers has uncovered how
hematopoietic stem cells (HSC) renew themselves.

HSCs refers to stem cells present in the blood and bone marrow that are used 
for  the replenishment of the body’s supply of blood and immune cells – 

  • in transplants for leukemia and myeloma.
  • Stem cells have the potential to transform into vital cells

    including muscle, bone, and blood vessels.

Understanding how HSCs form and renew themselves has potential application in the
treatment of

  • spinal cord injuries
  • degenerative disorders
  • diabetes.

Professor Peter Currie, of the Australian Regen Med Institute at Victoria’s Monash
University, led a research team to discover a crucial part of HSC’s development. Using 
a high-resolution microscopy, Prof. Curie’s team 

  • caught zebrafish embyonic SCs on film as they formed. 
  • the researchers were studying muscle mutations in the aquatic animal.

“Zebrafish make ESCs in exactly the same way as humans do, but their embryos and
larvae develop free living, but the larvae are both free swimming and transparent, so one could see every cell in the body forming, including ESCs,” explained Prof. Currie.

The researchers noticed in films that a

  •  ‘buddy cell’ came along to help the ESCs form.

Called endotome cells, 

  • they aided pre-ESCs to turn into ESCs.  

Prof. Currie said that endotome cells act as helper cells for pre-ESCs , 

  • helping them progress to become fully fledged stem cells.

The team not only

  • identified some of the cells and signals 
  • required for ESC formation, but also 
  • pinpointed the genes required 
  • for endotome formation in the first place.

The next step for the researchers is to 

  • locate the signals present in the endotome cells 
  • that trigger ESC formation in the embryo. 

This may provide clues for developing

  • specific blood cells on demand for blood-related disorders. 

Professor Currie also pointed out the discovery’s potential for 

  • correcting genetic defects in the cell and 
  • transplanting them back in the body to treat disorders.

The team’s work was published in the international journal Nature.


Jell-O Like Biomaterial Could Hold Key to Cancer Cell Destruction

by Estel Grace Masangkay

Scientists from Penn State University reported that a biomaterial made of tiny 
molecules was able to attract and destroy cancer cells.

Professor Yong Wang and bioengineering faculty at Penn State, built the 
tissue-like biomaterial to accomplish what chemotherapy could not –

  • kill every cancer cell without leaving
  • the possibility of a recurrence.

Prof. Wang and team built polymers 

  • from tiny molecules called monomers. They
  • then wove the polymers into 3D networks 

called hydrogels. Hydrogel is soft and flexible, 
like Jell-O, and it contains a lot of water, and

  • can be safely put into the body, unlike 

other implants that the body often tries 

  • to get rid of through the immune response.

“We want to make sure the materials we are using are compatible in the body.”

The researchers 

  • attached aptamers to the hydrogels, 
  • which release bio-chemical signal-only molecules 
  • that draw in cancer cells. 

Once attracted, the cancer cells are entrapped in the Jell-O-like substance. 

What happens next is 

  • an oligonucleotide binds to the protein-binding site of the aptamer 
  • and triggers the release of anticancer drugs at the proper time.

“Once we trap the cancer cells, we can deliver anticancer drugs 

  • to that specific location to kill them. 

This technique would help avoid the need for systemic medications that kill not only cancer cells, but normal cells as well. Systemic chemotherapy drugs

  • make patients devastatingly sick and possibly 
  • leave behind cancer cells to wreak havoc another day

If our new technique has any side effects at all, it would be only local side 
effects and not whole-body systemic side effects,” explained Prof. Wang.

The initial results of the research were published by Prof. Wang in the 
Journal of the American Chemical Society in 2012. Prof. Wang also shared 
the latest results of his work at the Society for Biomaterials Meeting &
 Exposition in April this year.


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